Method for the asymmetric synthesis of beta-lactone compounds

ABSTRACT

The present invention features methods of treating a cancer in a subject by administering an effective amount of a beta-lactone to the subject. The invention also features methods of inhibiting angiogenesis in a subject by administering an effective amount of an inhibitor of fatty acid synthase to the subject. These methods can be used to treat a variety of cancers and other diseases and conditions. The invention also features methods of identifying beta-lactones and other compounds that can be used in the methods of the invention for the treatment of tumors, inhibition of angiogenesis, and the treatment of diseases and conditions that involve pathological angiogenesis. The invention also features methods of synthesizing beta-lactones and features novel beta-lactone compounds.

PRIORITY APPLICATION INFORMATION

This application is a continuation-in-part of and claims priority toU.S. application Ser. No. 10/418,513, pending, filed Apr. 16, 2003, andwhich is hereby incorporated by reference in its entirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos. CA69036 and CA 106582 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to beta-lactones to inhibit theenzymatic activity of fatty acid synthase.

BACKGROUND OF THE INVENTION

Fatty acid synthase (FAS) is a multifunctional enzyme that catalyzes thesynthesis of long-chain fatty acids from small carbon substrates. Theenzyme contains six separate enzymatic pockets along with an acylcarrier protein, which act sequentially to perform repeatedcondensations of acetyl CoA and malonyl CoA, yielding predominantlypalmitate, a sixteen-carbon polyunsaturated fatty acid. Following sevensuch condensation cycles, palmitate remains covalently attached to theacyl carrier protein of the enzyme until it is liberated by the finalenzymatic pocket on the enzyme, the intrinsic thioesterase.

Fatty acid synthesis has long been thought unimportant in most normaltissues, and the enzyme is down-regulated by dietary lipid. However,increased FAS expression and activity in tumors is well documented.Tumor cell dependence on de novo fatty acid synthesis is viewed as ametabolic anomaly, with the endogenously-synthesized fatty acidsapparently incorporated into membrane phospholipids in preparation forcell division.

Given the prevalence of cancer that is refractory to current therapies,there is a need for new cancer treatment strategies.

Moreover, there is a need for new compounds useful for providing cancertherapy treatments.

SUMMARY OF THE INVENTION

Described herein are asymmetric beta-lactones compounds and methods fortheir synthesis.

The invention includes methods for the asymmetric synthesis ofbeta-lactone compounds comprising the steps of forming a ketene dimerfrom an acid chloride; and hydrogenating the ketene dimer to generate acis-beta-lactone.

In some embodiments the method is a one-pot synthesis method. In someembodiments the ketene dimer formed is isolated before the hydrogenationstep.

In some embodiments of the method the isolation of the ketene dimer isby silica gel purification.

In some embodiments of the method the acid chloride has the formula VIIwherein R comprises a hydrogen, an alkyl group, a cycloalkyl group, aheterocycloalkyl group, an alkoxy group, a hydroxyalkyl group, ahalogenated alkyl group, an alkoxyalkyl group, an alkenyl group, analkynyl group, an aryl group, a heteroaryl group, an aralkyl group, ahydroxy group, an amine group, an amide, an ester, a carbonate group, acarboxylic acid, an aldehyde, a keto group, an ether group, a halide, aurethane group, a silyl group or a sulfo-oxo group.

In some embodiments of the method the R comprises n-butyl, cyclopentyl,cyclohexyl, benzyl, CH.sub.2CO.sub.2Me or 11-methoxynonyl or n-butyl.

In some embodiments of the method the step of forming a ketene dimerfrom an acid chloride comprises using a catalyst wherein said catalystis selected from the group consisting of QND, O-TBS QND, O-TMS QND, andO-TMS QUIN.

In some embodiments of the method the step of hydrogenating the ketenedimer comprises using a palladium on carbon catalyst, which can be at aconcentration of about between 1 mol % and 5 mol %.

In some embodiments of the method the catalyst is accompanied by anamine which can be triethylamine.

In some embodiments of the method the step of hydrogenating a ketenedimer is performed for 30 minutes at 30 psi H.sub.2.

In some embodiments the method can further include an epimerizationstep.

In some embodiments of the method the epimerization step comprises thefurther steps of deprotonating the cis-beta lactone; and quenching thedeprotonated species under low temperature.

In some embodiments of the method the step of deprotonating is performedusing lithium hexamethyldisilazide (LiHMDS) or related bases such aslithium diisopropylamide (LDA), sodium hexamethyldisilazide (NaHMDS), orlithium tetramethylpiperidide (LiTMP).

In some embodiments of the method the step of quenching the deprotonatedspecies is performed using acetic acid.

In some embodiments the method may further include converting thecis-beta-lactone to a trisubstituted species. In some embodiments of themethod the converting step includes alkylation or acylation.

In some embodiments the method further includes enolization of thecis-beta-lactone; and addition of electrophiles.

In some embodiments of the method the enolization step is performedusing LDA, LiHMDS or NaHMDS.

In some embodiments of the method the electrophiles added to theenolated species are selected from the group consisting ofelectrophiles.

In some embodiments of the method the electrophile is a bulkyelectrophile or a smaller electrophile. In some embodiments the methodthe electrophile comprises CH₃, Benzyl or CO₂Me.

In some embodiments the invention is a compound synthesized by theinventive methods.

In some embodiments the invention is a compound of formula VI whereinR¹, R², R³, and R⁴ independently comprise a hydrogen, an alkyl group, acycloalkyl group, a heterocycloalkyl group, an alkoxy group, ahydroxyalkyl group, a halogenated alkyl group, an alkoxyalkyl group, analkenyl group, an alkynyl group, an aryl group, a heteroaryl group, anaralkyl group, a hydroxy group, an amine group, an amide, an ester, acarbonate group, a carboxylic acid, an aldehyde, a keto group, an ethergroup, a halide, a urethane group, a silyl group, a sulfo-oxo group, ora combination thereof, and wherein the stereochemistry at carbons a andb is R or S.

In some embodiments the invention is a compound of formula wherein Rcomprises n-butyl, cyclopentyl, cyclohexyl, benzyl, CH.sub.2CO.sub.2Meor 11-methoxynonyl.

In some embodiments the invention is a compound of formula VI wherein R1and R4 are n-butyl and R2 and R3 are hydrogen.

In some embodiments the invention is a compound of formula VI whereinthe compound is a compound selected from the group consisting of (3R,4S)-3-butyl-4-pentyloxetan-2-one, (3R, 4R)-3-butyl-4-pentyloxetan-2-one;(3R, 4S)-3-butyl-3-methyl-4-pentyloxetan-2-one; (3S,4S)-3-benzyl-3-butyl-4-pentyloxetan-2-one; and (3S,4S)-benzyl3-butyl-2-oxo-4-pentyloxetane-3-carboxylate.

In some embodiments the invention is a compound of formula VI whereinthe compound is synthesized using a method comprising the steps offorming a ketene dimer from an acid chloride; and hydrogenating theketene dimer to generate a cis-beta-lactone.

In some embodiments the invention is a compound of claim formula VIwherein said compound is synthesized using a method comprising the stepsof forming a ketene dimer from an acid chloride; hydrogenating theketene dimer to generate a cis-beta-lactone; and epimerization stepfurther comprising the steps of deprotonating the cis-beta lactone; andquenching the deprotonated species under low temperature.

In some embodiments the invention is a compound of claim formula whereinthe compound is synthesized using a method comprising the steps offorming a ketene dimer from an acid chloride; hydrogenating the ketenedimer to generate a cis-beta-lactone; and converting thecis-beta-lactone to a trisubstituted species.

In some embodiments the invention is a method of inhibiting a serinehydrolase comprising administering an effective amount of abeta-lactone.

In some embodiments of the methods the serine hydrolase is selected formthe group consisting of lipase, protease and esterase.

In some embodiments of the methods the serine hydrolase is fatty acidsynthase.

In some embodiments of the methods the beta-lactone comprises a formulaI compound wherein R¹, R², R³, and R⁴ are, independently, hydrogen, analkyl group, a cycloalkyl group, a heterocycloalkyl group, an alkoxygroup, a hydroxyalkyl group, a halogenated alkyl group, an alkoxyalkylgroup, an alkenyl group, an alkynyl group, an aryl group, a heteroarylgroup, an aralkyl group, a hydroxy group, an amine group, an amide, anester, a carbonate group, a carboxylic acid, an aldehyde, a keto group,an ether group, a halide, a urethane group, a silyl group, a sulfo-oxogroup, or a combination thereof, and wherein the stereochemistry atcarbons a and b is R or S.

In some embodiments of the methods the beta-lactone comprises a formulaVI compound wherein R¹, R², R³, and R⁴ independently comprise ahydrogen, an alkyl group, a cycloalkyl group, a heterocycloalkyl group,an alkoxy group, a hydroxyalkyl group, a halogenated alkyl group, analkoxyalkyl group, an alkenyl group, an alkynyl group, an aryl group, aheteroaryl group, an aralkyl group, a hydroxy group, an amine group, anamide, an ester, a carbonate group, a carboxylic acid, an aldehyde, aketo group, an ether group, a halide, a urethane group, a silyl group, asulfo-oxo group, or a combination thereof, and wherein thestereochemistry at carbons a and b is R or S.

In any of the above aspects of the invention, the beta-lactone can beany beta-lactone (or any combination of beta-lactones), for example, butnot limited to, a beta-lactone wherein R¹ is a straight-chain alkylgroup (e.g., but not limited to, tetrahydrolipstatin, ebelactone A, orebelactone B).

In any of the above aspects of the invention, the beta lactone can havethe formula I:

wherein R¹, R², R³, and R⁴ are, independently, hydrogen, an alkyl group,a cycloalkyl group, a heterocycloalkyl group, an alkoxy group, ahydroxyalkyl group, a halogenated alkyl group, an alkoxyalkyl group, analkenyl group, an alkynyl group, an aryl group, a heteroaryl group, anaralkyl group, a hydroxy group, an amine group, an amide, an ester, acarbonate group, a carboxylic acid, an aldehyde, a keto group, an ethergroup, a halide, a urethane group, a silyl group, a sulfo-oxo group, ora combination thereof, and wherein the stereochemistry at carbons a andb is R or S, as described herein.

Alternatively in the invention, the beta lactone can have the formulaVI:

wherein R¹, R², R³, and R⁴ are, independently, hydrogen, an alkyl group,a cycloalkyl group, a heterocycloalkyl group, an alkoxy group, ahydroxyalkyl group, a halogenated alkyl group, an alkoxyalkyl group, analkenyl group, an alkynyl group, an aryl group, a heteroaryl group, anaralkyl group, a hydroxy group, an amine group, an amide, an ester, acarbonate group, a carboxylic acid, an aldehyde, a keto group, an ethergroup, a halide, a urethane group, a silyl group, a sulfo-oxo group, ora combination thereof, and wherein the stereochemistry at carbons a andb is R or S, as described herein.

For example, in various embodiments of any of the above aspects of theinvention, R² and R⁴ are hydrogen. Moreover, in various embodiments ofany of the above aspects of the invention, R¹ is an alkyl group, forexample, a straight chain C₁ to C₂₀ alkyl group. In still otherembodiments, R³is an alkyl group comprising an ester group (for example,a straight chain C₃ to C₂₀ alkyl group); moreover, the ester group canfurther comprise an amide group. In yet other embodiments, R³is analkenyl group (for example, a straight chain C₃ to C₂₀ alkenyl group)comprising an ester group; moreover, the ester group can furthercomprise an amide group.

In still other embodiments of any of the above aspects of the invention,R² and R⁴ are hydrogen, the stereochemistry at carbon a is S, and thestereochemistry at carbon b is S; moreover, R¹ can be an alkyl group oran alkenyl group. For example, in some embodiments of any of the aboveaspects of the invention, R¹ is an alkyl group, R² and R⁴ are hydrogen,R³ is an alkyl group comprising an ester group, the stereochemistry atcarbon a is S, and the stereochemistry at carbon b is S. In otherembodiments of any of the above aspects of the invention, R¹ is an alkylgroup, R² and R⁴ are hydrogen, R³is an alkenyl group comprising an estergroup, the stereochemistry at carbon a is S, and the stereochemistry atcarbon b is S. For example, the compound can be tetrahydrolipstatin orlipstatin.

In yet other embodiments of any of the above aspects of the invention,R¹ and R⁴ are hydrogen, and R¹ is a C₁ to C₅ alkyl group. For example,the alkyl group can be methyl, ethyl, propyl, isopropyl, butyl,isobutyl, or tert-butyl.

In yet other embodiments of any of the above aspects of the invention,R³is a C₃ to C₂₀ alkenyl group comprising at least one hydroxyl group orat least one protected hydroxyl group. Moreover, the alkenyl group canfurther comprise a carbonyl group. For example, in some embodiments ofany of the above aspects of the invention, R¹ is an alkyl group (forexample, a methyl or ethyl group), R² and R⁴ are hydrogen, R³ is analkenyl group comprising at least one hydroxyl group or at least oneprotected hydroxyl group, the stereochemistry at carbon a is S, and thestereochemistry at carbon b is S.

In various embodiments of any of the above aspects of the invention, thecompound can be ebelactone A or ebelactone B.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be apparent from thedescription, or can be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a scan of an SDS-polyacrylamide gel that displays theserine hydrolase activity profile for normal and neoplastic prostateepithelial cells.

FIG. 2 shows a scan of an SDS-polyacrylamide gel (top panel) thatindicates that the labeling of fatty acid synthase (FAS) by FP-Bodipy isinhibited in tetrahydrolipstatin-treated cells, and a scan of a Westernblot (bottom panel) that shows that the FAS expression levels aresimilar in the various samples.

FIG. 3 shows a scan of an SDS-polyacrylamide gel that displays theserine hydrolase profile of normal and neoplastic mammary epithelialcells.

FIG. 4 shows a scan of an SDS-polyacrylamide gel from an experiment inwhich beta-lactones were screened for their ability to inhibit theactivity of serine hydrolases.

FIG. 5 is a graph showing the effect of orlistat on apoptosis of normaland neoplastic mammary epithelial cells.

FIG. 6 is a graph showing the induction of cell cycle arrest in mammarycarcinoma cells by orlistat.

FIG. 7 is an X-ray crystal structure (POV Chem rendering) ofbeta-lactone 3c.

FIG. 8 illustrates some of the cinchona alkaloids and derivatives 9-11employed in the ketene dimerization.

FIGS. 9 a and 9 b is a three-dimensional plot illustrating in situ IRspectroscopy (Reaction View) monitoring of: (a) ketene formation fromhydrocinnamyl chloride at 23.deg.C. followed by addition of methanolleading to methyl cinnamate; (b) the ketene dimerization ofhydrocinnamyl chloride mediated by quinuclidine hydrochloride (5 mol %)and EtNi—Pr2 (1.0 equiv).

FIG. 10 illustrates the inventive method for preparing beta-lactones andillustrates the general structures and the specific structuressynthesized using the inventive method.

FIG. 11 illustrates epimerization and acylation/alkylation of thebeta-lactones synthesized using the invention method. Furtherillustrated are the general structures and the specific structuressynthesized hereby.

FIG. 12 illustrates use of the current invention method to prepare betalactones that closely resemble tetrahydrolipstatin and panclicin D.

FIG. 13 is a dose-response curve from a fluorogenic assay illustratingthe inhibition of FAS TE by beta-lactone 3a from Table 2, entry 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the surprising discovery thatbeta-lactone compounds can be used to inhibit serine hydrolase enzymeactivity. Fatty acid synthase (FAS), an enzyme known to be up-regulatedin tumor cells, and which has been linked to tumor cell proliferation.As shown herein, treatment of tumor cells with beta-lactone compoundsinduces cell cycle arrest and/or apoptosis, thereby preventing tumorcell proliferation and/or survival.

Described herein are activity-based profiling studies of serinehydrolases, a large gene family encompassing lipases, proteases, andesterases. The objective was to identify serine hydrolases thatrepresent unique points for therapeutic intervention in cancer, using anactivity-based profiling assay to screen for small molecule antagonists.In this screening method, the active site of the serine hydrolases iscovalently tagged with a probe comprised of fluorophosphonate linked toa reporter (Liu et al. “Activity-based protein profiling: the serinehydrolases.” Proc. Natl. Acad. Sci. U.S.A 96:14694-9, 1999).

A fluorescently-tagged fluorophosphonate probe (FP-TAMRA) was used todefine the profile of serine hydrolases in carcinoma cells, and toidentify inhibitors of these enzymes from a small set of beta-lactones.Specifically, three beta-lactones were tested for the ability to blocklabeling, by FP-TAMRA, of serine hydrolases expressed by mammarycarcinoma and prostate carcinoma cells. These beta-lactones includedebelactones A and B, related natural products from actinomycetes(Umezawa et al. “Ebelactone, an inhibitor of esterase, produced byactinomycetes.” J. Antibiot. (Tokyo). 33:1594-1596, 1980; and Uotani etal. “Structural studies on ebelactone A and B, esterase inhibitorsproduced by actinomycetes.” J. Antibiot. (Tokyo). 35:1495-1499, 1982).Another beta-lactone that was tested was tetrahydrolipstatin, a compoundalso known as orlistat. Orlistat is a synthetic derivative of thenatural product lipstatin, produced by streptomyces. Like a majority ofthe naturally-occurring beta-lactones, orlistat possesses trans-betalactone stereochemistry. Orlistat is a drug that has been approved bythe Food and Drug Administration (FDA) for weight management in obesepatients (McNeely and Benfield. “Orlistat.” Drugs 56:241-9; discussion250, 1998). Its effectiveness is attributed to its ability to preventabsorption of dietary fat by virtue of its inhibition of pancreaticlipase in the gastrointestinal tract.

All beta-lactones tested inhibited the enzymatic activity of fatty acidsynthase (FAS), one of the prominent serine hydrolases identified in ourassay. Additional experiments revealed that, surprisingly, orlistatelicits cell cycle arrest in tumor cells at the G₁/S boundary, and, inmore differentiated tumor cells, apoptosis soon follows. Thesediscoveries reveal a surprising and unappreciated anti-tumor activityfor orlistat and other beta-lactone compounds. Accordingly, anybeta-lactone, such as (but not limited to) those described herein, canbe used in the methods of the invention to treat or prevent tumors thatexpress FAS. Such treatment inhibits tumor cell proliferation and/orinduces tumor cell death. Similarly, the methods of the invention can beused to treat or prevent any tumor that overexpresses FAS (e.g., tumorscontaining cells that express higher levels of FAS than their normalcounterparts, e.g., levels that are at least: 10%, 25%, 50%, 75%, 90%,2-fold, 3-fold, 5-fold or 10-fold higher than their non-tumor cellcounterparts).

In its FDA-approved formulation, orlistat is administered orally, andthe effects of the drug are largely confined to the gastrointestinaltract, where it inactivates pancreatic lipase. We have found thatorlistat blocks fatty acid synthase activity and induces apoptosis in anumber of colon cancer cell lines. Accordingly, orlistat can beadministered to patients identified as being in need of therapy for thetreatment of colon cancer. In addition, orlistat can be administeredprophylactically to patients who are identified as being at relativelyhigh risk for developing colon cancer (e.g., a patient who is inremission from colon cancer, a patient whose family has a higher thannormal rate of colon cancer, a patient who has one or more geneticmutations that increase the risk of developing colon cancer (e.g., amutation in the p53 gene), and/or a patient who has or who has had adisease or condition that increases the risk of developing coloncancer); such patients are readily identified by those of skill in theart.

For other tumors (for example, but not limited to, those of the mammaryand prostate gland), orlistat can be administered via a different route(e.g., intravenously), or in a different formulation. Those of ordinaryskill in the art will readily be able to identify patients who shouldreceive treatment according to the methods of the invention for acancer, or patients who should receive prophylactic treatment on accountof having a higher than normal risk of developing a cancer that can betreated according to the methods of the invention. For example, apatient who is in remission from cancer, a patient whose family has ahigher than normal rate of cancer, a patient who has one or more geneticmutations that increase the risk of developing a cancer (e.g., BRCA-1mutations indicate a higher than normal risk of breast cancer), apatient who has had a laboratory test or medical diagnostic procedurethat indicates a higher than normal risk of cancer (e.g., a high levelof prostate-specific antigen (PSA) indicates a higher than normal riskfor prostate cancer, and the presence of colon polyps, as revealed bycolonoscopy, indicates a risk for colon cancer), and/or a patient whohas or who has had a disease or condition that increases the risk ofdeveloping a cancer (e.g., women who have had breast cancer are in somecases considered by those of skill in the art to have a higher risk ofdeveloping endometrial cancer), can be considered as candidates forprophylactic treatment to decrease the likelihood of developing a cancerthat could be treated according to the methods of the invention.

The present invention also provides methods based upon a secondsurprising discovery, i.e., the discovery that FAS is expressed byendothelial cells (the cells that form blood vessels), and is necessaryfor the proliferation of endothelial cells and for angiogenesis.Angiogenesis, also known as neovascularization, is the process by whichendothelial cells infiltrate a tissue, remodel to form blood vessels,and ultimately deliver blood to the tissue. A wide range of physiologicand pathophysiologic processes require angiogenesis, includingdevelopment, adipogenesis, psoriasis, macular degeneration, and tumorgrowth and metastasis (see, e.g., Auerbach, W. and Auerbach, R.“Angiogenesis inhibition: a review.” Pharmacol. Ther. 63:265-311, 1994;Folkman, J. “Angiogenesis in cancer, vascular, rheumatoid and otherdisease.” Nat. Med. 1:27-31, 1995; and Creamer, J. D. and Barker, J. N.“Vascular proliferation and angiogenic factors in psoriasis.” Clin. Exp.Dermatol. 20:6-9, 1995).

The experiments described herein show that FAS is necessary for theproliferation of endothelial cells and for angiogenesis, and thatinhibition of FAS activity in endothelial cells inhibits mitogenesis andinduces cell cycle arrest. Accordingly, the present invention providesmethods for inhibiting angiogenesis and for treating and/or preventingcancers and other diseases that involve pathological angiogenesis, byadministering compounds that inhibit FAS activity, thereby inhibitingangiogenesis and treating and/or preventing the disease.

Inhibitors or antagonists of FAS activity can be used in the methods ofthe invention as anti-angiogenic factors to treat and/or prevent anydisease or condition that involves pathological angiogenesis (i.e.,angiogenesis that allows a disease or condition to initially develop, tobe maintained, or to worsen). Such diseases include, but are not limitedto: macular degeneration, diabetic retinopathy, arthritis, obesity,psoriasis, eczema, and scleroderma. In addition, administration of FASantagonists according to the methods of the invention can be used totreat and/or prevent blood vessel tumors, such as haemangiomas,angiosarcomas, and Kaposi's sarcoma.

Moreover, since tumors cannot grow beyond a volume of 2-3 mm³ withoutrecruiting an additional blood supply, administration of an FASantagonist to inhibit angiogenesis provides a universal strategy fortreating and/or preventing solid tumors that can otherwise exhibitwidely different phenotypes. Inhibition of angiogenesis by inhibitingFAS activity can also be used to prevent the metastatic spread ofcancer, as infiltration of a tumor by blood vessels provides a route fortumor cells to enter the blood circulation and metastasize.

Examples of tumors that can be treated by administration of a FASantagonist to inhibit tumor angiogenesis include, but are not limitedto: tumors of the brain or nervous system (e.g., neuroblastoma, glioma,and glioblastoma), sarcomas (e.g., osteosarcoma, rhabdomyosarcoma,Ewing's sarcoma, angiosarcoma, Kaposi's sarcoma), lymphoma, and multiplemyeloma. Other types of tumors that can be treated by administration ofa FAS antagonist (e.g., a beta-lactone) to inhibit tumor angiogenesisinclude (but are not limited to): leukemias and carcinomas, e.g.,carcinomas of the breast, prostate, ovary, endometrium, colon, stomach,liver, pancreas, esophagus, lung, oral mucosa, or skin.

Any beta-lactone, e.g., but not limited to, those examples describedherein, can be used in the methods of the invention to inhibitangiogenesis in patients and subjects who would benefit from suchinhibition. In addition, any other non-beta-lactone compound thatinhibits FAS activity can be used as an anti-angiogenic factor in themethods of the invention. For example, the fungal compound cerulenin andits artificial derivative, c75 (Kuhajda et al. “Synthesis and antitumoractivity of an inhibitor of fatty acid synthase.” Proc. Natl. Acad. Sci.U.S.A. 97:3450-3454, 2000) can be used in the methods of the inventionto inhibit FAS activity in order to inhibit angiogenesis and treat anydisease or condition that involves pathological angiogenesis.

Beta-Lactone Compounds

The compounds of the invention and useful in all of the methods of thepresent invention are generally referred to as beta-lactones.Beta-lactones possess the core structure

where a number of different groups can be substituted for one or morehydrogen atoms of the —CH₂CH₂— unit present in the ring. For example,beta-lactones useful in the present invention are represented by formulaI

wherein R¹, R², R³, and R⁴ are, independently, hydrogen, an alkyl group,a cycloalkyl group, a heterocycloalkyl group, an alkoxy group, ahydroxyalkyl group, a halogenated alkyl group, an alkoxyalkyl group, analkenyl group, an alkynyl group, an aryl group, a heteroaryl group, anaralkyl group, a hydroxy group, an amine group, an amide, an ester, acarbonate group, a carboxylic acid, an aldehyde, a keto group, an ethergroup, a halide, a urethane group, a silyl group, a sulfo-oxo group, ora combination thereof, and wherein the stereochemistry at carbons a andb is R or S.

Alternatively the beta lactones can have the formula VI:

wherein R¹, R², R³, and R⁴ are, independently, hydrogen, an alkyl group,a cycloalkyl group, a heterocycloalkyl group, an alkoxy group, ahydroxyalkyl group, a halogenated alkyl group, an alkoxyalkyl group, analkenyl group, an alkynyl group, an aryl group, a heteroaryl group, anaralkyl group, a hydroxy group, an amine group, an amide, an ester, acarbonate group, a carboxylic acid, an aldehyde, a keto group, an ethergroup, a halide, a urethane group, a silyl group, a sulfo-oxo group, ora combination thereof, and wherein the stereochemistry at carbons a andb is R or S, as described herein.

Variables such as R¹-R⁴ used throughout the application are the samevariables as previously defined unless stated to the contrary.

The term “alkyl group” is defined as a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl,octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like.

The term “alkenyl group” is defined as a hydrocarbon group of 2 to 24carbon atoms and structural formula containing at least onecarbon-carbon double bond.

The term “alkynyl group” is defined as a hydrocarbon group of 2 to 24carbon atoms and a structural formula containing at least onecarbon-carbon triple bond.

The term “halogenated alkyl group” is defined as an alkyl, alkenyl, oralkynyl group as defined above with one or more hydrogen atoms presenton these groups substituted with a halogen (F, Cl, Br, I).

The term “aryl group” is defined as any carbon-based aromatic groupincluding, but not limited to, benzene, naphthalene, etc. The term“aromatic” also includes “heteroaryl group,” which is defined as anaromatic group that has at least one heteroatom incorporated within thering of the aromatic group. Examples of heteroatoms include, but are notlimited to, nitrogen, oxygen, sulfur, and phosphorous. The aryl groupcan be substituted or unsubstituted. The aryl group can be substitutedwith one or more groups including, but not limited to, alkyl, alkynyl,alkenyl, aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy,carboxylic acid, or alkoxy.

The term “cycloalkyl group” is defined as a non-aromatic carbon-basedring composed of at least three carbon atoms. Examples of cycloalkylgroups include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, etc. The term “heterocycloalkyl group” is acycloalkyl group as defined above where at least one of the carbon atomsof the ring is substituted with a heteroatom such as, but not limitedto, nitrogen, oxygen, sulphur, or phosphorous.

The term “aralkyl” is defined as an aryl group having an alkyl, alkynyl,or alkenyl group as defined above attached to the aromatic group. Anexample of an aralkyl group is a benzyl group.

The term “hydroxyl group” is represented by the formula —OH. The term“alkoxy group” is represented by the formula —OR, where R can be analkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl,or heterocycloalkyl group described above.

The term “hydroxyalkyl group” is defined as an alkyl, alkenyl, alkynyl,aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl groupdescribed above that has at least one hydrogen atom substituted with ahydroxyl group. The term “alkoxyalkyl group” is defined as an alkyl,alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, orheterocycloalkyl group described above that has at least one hydrogenatom substituted with an alkoxy group described above.

The term “amine group” is represented by the formula —NRR′, where R andR′ can be, independently, hydrogen or an alkyl, alkenyl, alkynyl, aryl,aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl groupdescribed above.

The term “amide group” is represented by the formula —C(O)NRR′, where Rand R′ can be an alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl,halogenated alkyl, or heterocycloalkyl group described above.

The term “ester” is represented by the formula —OC(O)R, where R can bean alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenatedalkyl, or heterocycloalkyl group described above.

The term “carbonate group” is represented by the formula —OC(O)OR, whereR can be an alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl,halogenated alkyl, or heterocycloalkyl group described above.

The term “carboxylic acid” is represented by the formula —C(O)OH.

The term “aldehyde” is represented by the formula —C(O)H.

The term “keto group” is represented by the formula —C(O)R, where R isan alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenatedalkyl, or heterocycloalkyl group described above.

The term “carbonyl group” is represented by the formula C═O.

The term “ether group” is represented by the formula R(O)R′, where R andR′ can be, independently, an alkyl, alkenyl, alkynyl, aryl, aralkyl,cycloalkyl, halogenated alkyl, or heterocycloalkyl group describedabove.

The term “halide” is defined as F, Cl, Br, or I.

The term “urethane” is represented by the formula —OC(O)NRR′, where Rand R′ can be, independently, an alkyl, alkenyl, alkynyl, aryl, aralkyl,cycloalkyl, halogenated alkyl, or heterocycloalkyl group describedabove.

The term “silyl group” is represented by the formula —SiRR′R″, where R,R′, and R″ can be, independently, an alkyl, alkenyl, alkynyl, aryl,aralkyl, cycloalkyl, halogenated alkyl, alkoxy, or heterocycloalkylgroup described above.

The term “sulfo-oxo group” is represented by the formulas —S(O)₂R,—OS(O)₂R, or, —OS(O)₂OR, where R can be an alkyl, alkenyl, alkynyl,aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl groupdescribed above.

R¹-R⁴ can, independently, possess two or more of the groups listedabove. For example, if R¹ is a straight chain alkyl group, one of thehydrogen atoms of the alkyl group can be substituted with a hydroxylgroup, an alkoxy group, etc. Depending upon the groups that areselected, a first group may be incorporated within the second group or,alternatively, the first group may be pendant (i.e., attached) to thesecond group. For example, with the phrase “an alkyl group furtherincluding an ester group,” the ester group may be incorporated withinthe backbone of alkyl group. Alternatively, the ester can be attached tothe backbone of the alkyl group. The nature of the group(s) that is(are) selected will determine if the first group is embedded or attachedto the second group.

The compounds represented by formula I can be optically active orracemic. The stereochemistry at carbons a and b can vary, and willdepend upon the spatial relationship between R¹, R², R³, and R⁴ to oneanother. In one embodiment, the stereochemistry at carbons a and b is S.In another embodiment, the stereochemistry at carbons a and b is R. In afurther embodiment, the stereochemistry at carbon a is S and thestereochemistry at carbon b is R. In a further embodiment, thestereochemistry at carbons a is R and the stereochemistry at carbon b isS. Using techniques known in the art, it is possible to vary thestereochemistry at carbons a and b.

In one embodiment, R¹ is an alkyl group. The alkyl group can be branchedor straight chain. In one embodiment, R¹ is a straight chain C₁ to C₂₀,C₃ to C₁₈, C₅ to C₁₆, C₇ to C₁₄, or C₉ to C₁₂ alkyl group. In anotherembodiment, R¹ is a C₁ to C₅ alkyl group, such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, or tert-butyl.

In one embodiment, R³ is a branched or straight chain C₃ to C₂₀, C₅ toC₁₈, C₇ to C₁₆, C₉ to C₁₄, or C₁₀ to C₁₂ alkyl group. Alternatively, R³is a branched or straight chain C₃ to C₂₀, C₅ to C₁₈, C₇ to C₁₆, C₉ toC₁₄, or C₁₀ to C₁₂ alkenyl group. The alkyl or alkenyl group of R³ canbe substituted with one or more of the following groups: an alkyl group,a cycloalkyl group, a heterocycloalkyl group, an alkoxy group, ahydroxyalkyl group, a halogenated alkyl group, an alkoxyalkyl group, analkenyl group, an alkynyl group, an aryl group, a heteroaryl group, anaralkyl group, a hydroxy group, an amine group, an amide, an ester, acarbonate group, a carboxylic acid, an aldehyde, a keto group, an ethergroup, a halide, a urethane group, a silyl group, or a sulfo-oxo group.In a further embodiment, when R³ is an alkyl or alkenyl group, the alkylor alkenyl group comprises an ester group. The ester can optionallycomprise any of the groups listed above. In one embodiment, the estergroup further comprises an amide group.

In another embodiment, R³ is a C₃ to C₂₀ alkenyl group comprising atleast one hydroxyl group or at least one protected hydroxyl group. Theterm “protected hydroxyl group” refers to a hydroxyl group that has beenconverted to a group such as, but not limited to, an alkoxy group, anester group, an aldehyde, a keto group, a carbonate, an amide, a silylgroup, or a sulfo-oxo group. “Protecting Groups in Organic Synthesis” byT. W. Green, John Wiley and Sons, 1981, pp. 10-81, which is incorporatedby reference, discloses numerous techniques for protecting a hydroxylgroup. In another embodiment, when R³ is an alkenyl group comprising atleast one hydroxyl group or at least one protected hydroxyl group, R³further comprises a carbonyl group.

In one embodiment, R¹ is an alkyl group, R² and R⁴ are hydrogen, R³ isan alkyl group comprising an ester group, the stereochemistry at carbona is S, and the stereochemistry at carbon b is S.

In another embodiment, R¹ is an alkyl group, R² and R⁴ are hydrogen, R³is an alkenyl group comprising an ester group, the stereochemistry atcarbon a is S, and the stereochemistry at carbon b is S.

In another embodiment, R¹ is an alkyl group, R² and R⁴ are hydrogen, R³is an alkenyl group comprising at least one hydroxyl group or at leastone protected hydroxyl group, the stereochemistry at carbon a is S, andthe stereochemistry at carbon b is S.

In other embodiments, the beta-lactone is a compound provided below,which can be prepared using techniques known in the art:

-   (1S)-1-[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]tridecyl ester L-leucine-   1-[(3-hexyl-4-oxo-2-oxetanyl)methyl]dodecyl ester L-leucine-   3-hexyl-4-(2-hydroxytridecyl)-2-oxetanone-   (3S,4S)-3-hexyl-4-[(1S)-1-hydroxytridecyl]-2-oxetanone-   1-[(3-hexyl-4-oxo-2-oxetanyl)methyl]dodecyl ester    N-[(1,1-dimethylethoxy)carbonyl]-L-leucine-   1-[(3-hexyl-4-oxo-2-oxetanyl)methyl]dodecyl ester    N-benzoyl-L-Leucine-   3-hexyl-4-(2-hydroxytridecyl)-2-oxetanone-   (2S,3R)-4-oxo-3-(4-pentenyl)-2-oxetanecarboxylic acid    1,1-dimethylethyl ester-   3-hexyl-4-[2-(3-hydroxypropoxy)tridecyl]-2-oxetanone-   4-[2-(3-chloropropoxy)tridecyl]-3-hexyl-2-oxetanone-   1-[(3-hexyl-4-oxo-2-oxetanyl)methyl]dodecyl ester    N-[4-(hydroxymethyl)benzoyl]-L-Leucine-   (2E,4E,7R)-11-[(2R,3R)-3-(hydroxymethyl)-4-oxo-2-oxetanyl]-3,5,7-trimethyl-2,4-undecadienal-   (3R,4R)-3-(phenylmethyl)-4-[(trimethylsilyl)ethynyl]-2-oxetanone-   (3R,4R)-4-[4-[(4-methoxyphenyl)methoxyl]-1-butynyl]-3-methyl-2-oxetanone-   (3R,4R)-3-methyl-4-[3-(phenylmethoxy)-1-propynyl]-2-oxetanone-   L-alanyl-3-[(1R,2S)-2-[[[(2R,3S)-3-[(1S)-methylpropyl]-4-oxo-2-oxetanyl]carbonyl]amino]cyclopropyl]-L-alanine-   L-alanyl-N5-[[(2R,3S)-3-[(1S)-1-methylpropyl]-4-oxo-2-oxetanyl]carbonyl]-L-ornithine-   N-[(1,1-dimethylethoxy)carbonyl]-L-alanyl-3-[(1R,2S)-2-[[[(2R,3S)-3-[(1S)-methylpropyl]-4-oxo-2-oxetanyl]carbonyl]amino]cyclopropyl]-L-alanine

(1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]methyl]hexyl esterN-formyl-L-valine

-   6-[(2-amino-5-chlorobenzoyl)amino]-2,4,6,7-tetradeoxy-2,4,4-trimethyl-,.beta.-lactone,    L-ribo-5-Heptulosonic acid-   6-[[2-(acetylamino)-5-chlorobenzoyl]amino]-2,4,6,7-tetradeoxy-2,4,4-trimethyl-L-ribo-5-heptulosonic    acid beta-lactone-   [3S-[3.alpha., 4.beta.(1R*, 3E, 5S*, 7R*, 8S*,    9S*)]]-4-[8-(acetyloxy)-1,3,5,7,9-pentamethyl-6-oxo-3-undecenyl]-3-methyl-2-oxetanone-   [3S-[3.alpha., 4.beta.(1R*, 3E, 5S*, 7R*, 8S*,    9S*)]]-4-[8-(acetyloxy)-1,3,5,7,9-pentamethyl-6-oxo-3-undeceny]-3-ethyl-2-oxetanone-   4-(8-hydroxy-1,3,5,7,9-pentamethyl-6-oxoundecyl)-3-methyl-2-oxetanone-   (3S,4S)-3-hexyl-4-[(2R)-2-hydroxytridecyl1]-2-oxetanone-   (3S,4S)-3-hexyl-4-[(2R)-2-[[tris(1-methylethyl)silyl]oxy]tridecyl]-2-oxetanone-   (3S,4S)-3-(2-hexenyl)-4-[(2-undecyl-1,3-dioxolan-2-yl)methyl]-2-oxetanone-   (3S,4S)-3-(2-hexenyl)-4-(2-oxotridecyl)-2-oxetanone-   (4S)-3-3-di-2-hexenyl-4-[(2-undecyl-1,3-dioxolan-2-yl)methyl]-2-oxetanone-   L-alanyl-3-[(1R,2S)-2-[[[(2R,3S)-3-[(1S)-1-methylpropyl]-4-oxo-2-oxetanyl]carbonyl]amino]cyclopropyl]-L-alanine-   (1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]methyl]dodecyl ester    N-[(phenylmethoxy)carbonyl]-L-leucine-   (3S,4S)-3-hexyl-4-[(2S)-2-[tetrahydro-2H-pyran-2-yl)oxy]tridecyl]-2-oxetanone-   4-nonyl-3-[8-(phenylmethoxy)octyl]-2-oxetanone-   3-(8-hydroxyoctyl)-4-nonyl-2-oxetanone-   (2E,4E,7R)-11-[(2R,3R)-3-ethenyl-4-oxo-2-oxetanyl]-3,5,7-trimethyl-2,4-Undecadienoic    acid diphenylmethyl ester-   (2E)-3-[3-[(2R)-6-[(2R,3R)-3-ethenyl-4-oxo-2-oxetanyl]-2-methylhexyl]-3-methyloxiranyl]-2-butenoic    acid diphenylmethyl ester-   (2E)-3-[3-[(2R)-6-[(2R,3R)-3-(hydroxymethyl)-4-oxo-2-oxetanyl]-2-methylhexyl]-3-methyloxiranyl)-2-butenoic    acid diphenylmethyl ester-   (2E,4E,7R)-11-[(2R,3R)-3-(hydroxymethyl)-4-oxo-2-oxetanyl]-3,5,7-trimethyl-2,4-undecadienoic    acid diphenylmethyl ester-   (1S)-1-[[(2S,3S)-3-decyl-4-oxo-2-oxetanyl]methyl]octyl ester    N-formyl-glycine-   (3S,4S)-4-[(2R)-2-hydroxynonyl]-3-(8-methylnonyl)-3-(trimethylsilyl)-2-oxetanone-   (3S,4S)-,3-dodecyl-4-[(2R)-2-hydroxynonyl]-3-(trimethylsilyl)-2-oxetanone-   (3R,4S)-3-decyl-4-[(2R)-2-hydroxynonyl]-2-oxetanone-   [3S-[3.alpha.,4.beta.(S*)]]-3-decyl-4-(2-hydroxynonyl)-2-oxetanone-   (3R,4S)-4-[(2R)-2-[[(1,1-dimethylethyl)dimethylsilyl]oxy]nonyl]-3-(8-methylnonyl)-3-(trimethylsilyl)-2-oxetanone-   (3S,4S)-4-[(2R)-2-[[(1,1-dimethylethyl)dimethylsilyl]oxy]nonyl]-3-(8-methylnonyl)-3-(trimethylsilyl)-2-oxetanone-   (3R,4S)-4-[(2R)-2-hydroxynonyl]-3-(8-methylnonyl)-3-(trimethylsilyl)-2-oxetanone-   (3S,4S)-4-[(2R)-2-hydroxynonyl]-3-(8-methylnonyl)-2-oxetanone-   (1S)-1-[[(2S,3S)-3-(8-methylnonyl)-4-oxo-2-oxetanyl]methyl]octyl    ester N-(triphenylmethyl)-L-alanine-   (3R,4S)-3-decyl-4-[(2R)-2-[[(1,1-dimethylethyl)dimethylsilyl]oxy]nony]-3-(trimethylsilyl)-2-oxetanone-   (3S,4S)-3-decyl-4-[(2R)-2-[[(1,1-dimethylethyl)dimethylsilyl]oxy]nony]-3-(trimethylsilyl)-2-oxetanone-   [3S-[3.alpha.,4.beta.(S*)]]-3-decyl-4-(2-hydroxynonyl)-2-oxetanone-   (3S-[3.alpha.,4.beta.(S*)]]-3-decyl-4-[2-[[(1,1-dimethylethyl)dimethylsilyl]oxy]nonyl]-2-oxetanone-   [3S-[3-.alpha.,4.beta.(S *)]]-3-decyl-4-(2-hydroxynonyl)-2-oxetanone-   (3R,4R)-3-methyl-4-(2-phenylethyl)-2-oxetanone-   4-heptyl-3,3-dimethyl-2-oxetanone-   trans-4-cyclohexyl-3,4-dimethyl-2-oxetanone-   [2R-[2.alpha.(2E,4E),3.beta.]]-11-[3-(hydroxymethyl)-4-oxo-2-oxetanyl]-3,5,7-trimethyl-2,4-undecadienoic    acid methyl ester-   (1S)-1-[[(2S,3S)-3-(8-methylnonyl)-4-oxo-2-oxetanyl]methyl]octyl    ester N-formyl-glycine-   (1S)-1-[[(2S,3S)-3-decyl-4-oxo-2-oxetanyl]methyl]octyl ester    N-formyl-glycine-   (1S)-1-[[(2S,3S)-3-decyl-4-oxo-2-oxetanyl]methyl]octyl ester    N-formyl-L-alanine-   [3R-[3.alpha.4.beta(*)]]-3-[[[(1,1,    -dimethylethyl)diphenylsilyl]oxy]methyl]-4-(5-methyl-7-oxooctyl)-2-oxetanone-   trans-4-oxo-3-[(triphenylmethoxy)methyl]-2-oxetaneundecanoic acid    methyl ester-   trans-3-(hydroxymethyl)-4-oxo-2-oxetaneundecanoic acid-   (2S-trans)-N,N-diethyl-3-hexyl-4-oxo-2-oxetanepentanamide-   N-formyl-,4-(3-hexyl-4-oxo-2-oxetanyl)butyl ester, (2S-trans)-,    L-Leucine-   (3S-trans)-3-hexyl-4-(4-hydroxybutyl)-2-oxetanone-   [2R-[2.alpha.(2E,4E,7R*),3.beta.]]-3,5,7-trimethyl-11-[4-oxo-3-[(triphenylmethoxy)methyl]-2-oxetanyl]-2,4-undecadienoic    acid methyl ester-   [3.alpha.(E),4.alpha.]-3-(1,3-butadienyl)-3-methyl-4-pentyl-2-oxetanone-   [3.alpha.(E),4.beta.]-3-(1,3-butadienyl)-3-methyl-4-pentyl-2-oxetanone-   [2R-[2.alpha.(2E,4E,7R*),3.beta]]-11-[3-(methoxymethyl)-4-oxo-2-oxetanyl]-3,5,7-trimethyl-2,4-undecadienoic    acid methyl ester-   [2R-[2.alpha.(2E,4E),3.beta.]]-11-[3-(hydroxymethyl)-4-oxo-2-oxetany    ]-3,5,7-trimethyl-2,4-undecadienoic acid methyl ester-   [3.alpha.,4.beta.(1R*,3E,5S    *,7R*,8R*)]-4-[8-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-1,3,5,7-tetramethyl-9-methylene-6-oxo-3-undecenyl]-3-methyl-2-oxetanone-   [3.alpha.,4.beta.(1R*,3E,5S*,7R*,8R*)]-4-[8-hydroxy-1,3,5,7-tetramethyl-9-methylene-6-oxo-3-undecenyl]-3-methyl-2-oxetanone-   [3.alpha.,4.beta.(1R*,3E,5S*,7R*,8S*,9R*)]-4-[8-hydroxy-1,3,5,7,9-pentamethyl-6-oxo-3-undecenyl)-3-methyl-2-oxetanone-   1-[(3-fluoro-3-hexyl-4-oxo-2-oxetanyl)methyl]dodecyl ester    N-formyl-L-leucine-   [2S-[2.alpha.(R*),3.alpha.]]-1-[(3-fluoro-3-hexyl-4-oxo-2-oxetanyl)methyl]dodecyl    ester N-formyl-L-leucine-   [3R-[3.alpha.,4.beta.(R*)]]-3-(hydroxymethyl)-4-(5-methyl-7-oxooctyl)-2-oxetanone-   [3S-[3.alpha.,4.beta.(2S    *,5Z)]]-3-hexyl-4-(2-hydroxy-5-tridecenyl)-2-oxetanone-   4-[8-(acetyloxy)octyl]-3-(1-methyl-2-propenyl)-2-oxetanone-   (3R,4S)-3-butyl-4-pentyloxetan-2-one-   (3R,4R)-3-butyl-4-pentyloxetan-2-one-   (3R,4S)-3-butyl-3-methyl-4-pentyloxetan-2-one-   (3S,4S)-3-benzyl-3-butyl-4-pentyloxetan-2-one-   (3S,4S)-benzyl 3-butyl-2-oxo-4-pentyloxetane-3-carboxylate    As well as the compounds shown in FIGS. 10, 11 and 12.

In another embodiment, the beta-lactones disclosed in InternationalPublication No. WO 200004300, Japanese Publication No. 03115274,European Publication No. 185359 A2, and U.S. Pat. Nos. 4,931,463,5,175,186, 5,246,960, 4,873,260, 4,806,564, 4,983,746, 5,260,310,5,376,674, 5,466,708, and 5,399,720, which are incorporated by referencein their entireties, are useful in the invention.

In one embodiment, the compound having the formula I istetrahydrolipstatin, which is also referred to as orlistat. Thestructure of tetrahydrolipstatin is depicted in formula II. In anotherembodiment, the compound is lipstatin, which is depicted in formula III.The synthesis of tetrahydrolipstatin and lipstatin is disclosed in U.S.Pat. No. 4,598,089, which is incorporated by reference.

In another embodiment, the compound having the formula I is ebelactone Aand B, which are depicted in formulas IV and V, respectively. Thesynthesis of ebelactone A and B is disclosed in the journal article byPaterson and Hulme entitled “Total Synthesis of (−)-Ebelactone A and B”J. Org. Chem., 1995, 60(11), 3288-3300, which is incorporated byreference in its entirety.

Methods of Administration of Beta-Lactones and Other Inhibitors of FattyAcid Synthase Activity

The FAS antagonists for use in the methods of the invention can beadministered to subjects with a pharmaceutically acceptable diluent,carrier, or excipient, in unit dosage form. By “pharmaceuticallyacceptable” is meant a material that is not biologically or otherwiseundesirable, i.e., the material may be administered to an individualalong with an FAS antagonist without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of thecomponents of the pharmaceutical composition in which it is contained.Conventional pharmaceutical practice may be employed to provide suitableformulations or compositions to administer such compositions tosubjects. Any appropriate route of administration may be employed, forexample, but not limited to, intravenous, parenteral, transcutaneous,subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic,intraventricular, intracapsular, intraspinal, intracisternal,intraperitoneal, intranasal, intrarectal, intravaginal, aerosol, or oraladministration. Therapeutic formulations may be in the form of liquidsolutions or suspensions; for oral administration, formulations may bein the form of tablets or capsules; for intranasal formulations, in theform of powders, nasal drops, or aerosols; for intravaginalformulations, vaginal creams, suppositories, or pessaries; fortransdermal formulations, in the form of creams or distributed ontopatches to be applied to the skin.

Methods well known in the art for making formulations are found in, forexample, Remington: The Science and Practice of Pharmacy (19th ed.) ed.A. R. Gennaro, Mack Publishing Company, Easton, Pa. 1995. Formulationsfor parenteral administration may, for example, contain excipients,sterile water, or saline, polyalkylene glycols such as polyethyleneglycol, oils of vegetable origin, or hydrogenated napthalenes.Biocompatible, biodegradable lactide polymer, lactide/glycolidecopolymer, or polyoxyethylene-polyoxypropylene copolymers may be used tocontrol the release of the compounds. Other potentially usefulparenteral delivery systems for molecules of the invention includeethylene-vinyl acetate copolymer particles, osmotic pumps, implantableinfusion systems, and liposomes. Formulations for inhalation may containexcipients, for example, lactose, or may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or may be oily solutions for administration in theform of nasal drops, or as a gel.

Any beta-lactone or other compound of the invention can be administeredsingly or in combination. In just example, tetrahydrolipostatin can beadministered by itself or in combination with ebelactone A and/or B,and/or in combination with another compound that inhibits FAS.

Dosage

The beta-lactones and other FAS antagonists for use in the methods ofthe invention may be administered to a subject in an effective amount,i.e., amount sufficient to partially or fully inhibit FAS activity in asubject in need thereof, e.g., to treat a cancer or to inhibitangiogenesis in a subject in need of such treatment. One of ordinaryskill in the art will understand that the optimal dosage used will varyaccording to the individual being treated and the particular cancer,disease, or other condition for which the individual is being treated,the particular compound being used, and the chosen route ofadministration. The optimal dosage will also vary among individuals onthe basis of age, size, weight, gender, and physical condition. Methodsfor determining optimal dosages are described, for example, inRemington: The Science and Practice of Pharmacy (19th ed.) ed. A. R.Gennaro, Mack Publishing Company, Easton, Pa. 1995. Typically, apharmaceutically effective dosage would be between about 0.001 and 200mg/kg body weight of the subject to be treated.

Efficacy

The efficacy of administration of a particular dose of a beta-lactone orother FAS antagonist according to the methods of the invention can bedetermined by evaluating the particular aspects of the medical history,signs, symptoms, and objective laboratory tests that are known to beuseful in evaluating the status of a subject in need of inhibition ofFAS for the treatment of cancer or other diseases and/or conditions.These signs, symptoms, and objective laboratory tests will vary,depending upon the particular disease or condition being treated orprevented, as will be known to any clinician who treats such patients ora researcher conducting experimentation in this field. For example, if,based on a comparison with an appropriate control group and/or knowledgeof the normal progression of the disease in the general population orthe particular individual: 1) a subject's physical condition is shown tobe improved (e.g., a tumor has partially or fully regressed), 2) theprogression of the disease or condition is shown to be stabilized, orslowed, or reversed, or 3) the need for other medications for treatingthe disease or condition is lessened or obviated, then a particulartreatment regimen will be considered efficacious.

Identification of Compounds That Inhibit FAS Activity

In general, compounds that inhibit the activity of FAS (i.e.,beta-lactones and other FAS antagonists) can be identified fromlibraries of natural products or synthetic (or semi-synthetic) extractsor chemical libraries according to methods known in the art and/ordescribed herein. Such screening methods include (but are not limitedto): serine hydrolase activity-profiling assays, [¹⁴C]-acetateincorporation assays, cell proliferation assays, apoptosis assays,and/or angiogenesis assays (see, e.g., Salcedo et al. “Human endothelialcells express CCR2 and respond to MCP-1: direct role of MCP-1 inangiogenesis and tumor progression.” Blood 96:34, 40, 2000). Thoseskilled in the field of drug discovery and development will understandthat the precise source of test extracts or compounds is not critical tothe screening procedure(s) of the invention. Accordingly, virtually anynumber of chemical extracts or compounds can be screened using theexemplary methods described herein. Examples of such extracts orcompounds include, but are not limited to, plant-, fungal-, prokaryotic-or animal-based extracts, fermentation broths, and synthetic compounds,as well as modification of existing compounds. Numerous methods are alsoavailable for generating random or directed synthesis (e.g.,semi-synthesis or total synthesis) of any number of chemical compounds,including, but not limited to, saccharide-, lipid-, peptide-, andnucleic acid-based compounds (e.g., but not limited to, antibodies,peptides, and aptamers). Synthetic compound libraries are commerciallyavailable, e.g., from Brandon Associates (Merrimack, N.H.) and AldrichChemical (Milwaukee, Wis.).

Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant, and animal extracts are commercially available from anumber of sources, including Biotics (Sussex, UK), Xenova (Slough, UK),Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar,U.S.A. (Cambridge, Mass.). In addition, natural and syntheticallyproduced libraries are generated, if desired, according to methods knownin the art, e.g., by standard extraction and fractionation methods.Furthermore, if desired, any library or compound is readily modifiedusing standard chemical, physical, or biochemical methods.

In sum, a test compound for use in the assay methods of the inventioncan be any molecule, be it naturally-occurring or artificially-derived,that is surveyed for its ability to: inhibit the activity of fatty acidsynthase, inhibit angiogenesis or a disease that involves pathogenicangiogenesis, inhibit cell proliferation, promote apoptosis, and/orpromote cell cycle arrest.

Samples for use in the assay methods of the invention include anyspecimen that can be tested for fatty acid synthase activity and/or thatcan be used to identify compounds that inhibit fatty acid synthase,inhibit angiogenesis or a disease that involves pathogenic angiogenesis,inhibit cell proliferation, promote apoptosis, and/or promote cell cyclearrest. Examples include, but are not limited to: a sample from apatient or subject, such as a cell, tissue, or tumor sample; a cell(e.g., a prokaryotic or eukaryotic cell that expresses endogenous orrecombinant FAS); a lysate (or lysate fraction) or extract derived froma cell; or a molecule derived from a cell or cellular material.

Those skilled in the art of drug discovery and development readilyunderstand that methods for de-replication (e.g., taxonomicde-replication, biological de-replication, and chemical de-replication,or any combination thereof) or the elimination of replicates or repeatsof materials already known for their effect on FAS should be employedwhenever possible.

When a crude extract is found to have a desired activity, furtherfractionation of the positive lead extract is necessary to isolatechemical constituents responsible for the observed effect. Thus, thegoal of the extraction, fractionation, and purification process is thecareful characterization and identification of a chemical entity withinthe crude extract that inhibits FAS activity. The same assays for thedetection of activities in mixtures of compounds can be used to purifythe active component and to test derivatives thereof. Methods offractionation and purification of such heterogenous extracts are knownin the art. If desired, compounds shown to be useful agents fortreatment are chemically modified according to methods known in the art.Compounds identified as being of therapeutic value may be subsequentlyanalyzed using in vitro assays (e.g., for cell cycle arrest, apoptosis,and/or angiogenesis) and in vivo animal models for diseases, conditions,and/or biological processes (e.g., cancer, angiogenesis, or a diseaseinvolving pathological angiogenesis) in which it is desirable to inhibitFAS activity to treat or prevent the disease or condition.

EXAMPLES

The present invention is more particularly described in the followingexamples, which are intended as illustrative only since numerousmodifications and variations thereof will be apparent to those ofordinary skill in the art.

Example I Orlistat and Ebelactones A and B Inhibit Fatty Acid SynthaseActivity and Induce Selective Apoptosis of Prostate Tumor Cells

Recent work in the area of chemical biology points the way toward directprofiling of protein activity. Two groups have shown it possible tocreate chemical probes that react at the active site of multiple enzymesof a given class. Liu et al showed how to obtain the profile of serinehydrolase activity with a probe containing fluorophosphonate as thewarhead and biotin as the reporter (Liu et al. “Activity-based proteinprofiling: the serine hydrolase.” Proc. Natl. Acad. Sci. U.S.A96:14694-9, 1999). In a related approach, Bogyo's group showed that thefamily of cysteine proteinases could be tagged covalently with reactiveepoxides (Greenbaum et al. “Epoxide electrophiles as activity-dependentcysteine protease profiling and discovery tools” Chem. Biol. 7:569-81,2000). Because activity-based probes bind at the active site of anenzyme, a direct measure of the level of active enzyme can be obtained,and it becomes possible to use straightforward competition assays toscreen for inhibitors.

The present study employs such an approach. An activity-based profilingeffort was coupled to a simultaneous screen for antagonists of serinehydrolases in prostate cancer cells. This was accomplished with anactivity-based probe comprised of a fluorophosphonate warhead linked tothe tetramethyl rhodamine fluorophore (FP-TAMRA) (Patricelli et al.“Direct visualization of serine hydrolase activities in complexproteomes using fluorescent active site-directed probes.” Proteomics 1:1067-1071, 2001).

FIG. 1 is a scan of an SDS-polyacrylamide gel showing an activityprofiling experiment for normal and neoplastic prostate epithelialcells. Lysates were generated from primary cultures of normal prostaticepithelial cells (PrEC), and from three prostate tumor cell lines(LNCaP, DU-145, and PC-3). Lysates (40 μg of total protein, at aconcentration of 1 mg/ml) were incubated with FP-PEG-TAMRA (2 μM) forone hour at room temperature. Reactions were stopped by the addition ofSDS-PAGE loading buffer containing β-mercaptoethanol and boiling.Non-specific labeling with the probe activity was measured in samplesthat were boiled prior to the addition of fp-PEG-TAMRA (lanes marked +).Samples were resolved by 10% SDS-PAGE and visualized at 605 nm using aHitachi flat bed gel scanner (lanes 1-8). The effect of threebeta-lactones on the activity-labeling of serine hydrolases from PC-3cells was assessed in a similar manner. Prior to incubation withFP-PEG-TAMRA, lysates (40 μg) were pre-incubated for 30 min with 100 μMof ebelactone A (lane 10), ebelactone B (lane 11), or orlistat (lane12). Following labeling with FP-PEG-TAMRA the reactions were halted andenzyme activity visualized as described above.

For each cell line tested as described above, approximately fifteendifferent hydrolases were visualized as fluorescent bands on SDS gels.The pattern of serine hydrolase expression was generally similar amongthe cell lines, with two significant distinctions. A hydrolase with amass of approximately 270 kDa was expressed in all of the tumor lines,but was absent in normal PrECs. Peptide mass fingerprinting with massspectrometry showed this band to be fatty acid synthase (FAS), anobservation that was confirmed by immunoprecipitating the complexbetween FP-TAMRA and FAS.

As the single eukaryotic enzyme capable of synthesizing palmitate, FASis responsible for generating the precursor for the majority of cellularfatty acids. FAS has a unique structure and mode of action. The enzymecontains six separate enzymatic pockets along with an acyl carrierprotein. Palmitate is generated by the enzymes repeated condensation ofacetyl co-A and malonyl co-A. Seven such condensation cycles yield thesixteen-carbon polyunsaturated fatty acid palmitate. Palmitate remainscovalently attached to the acyl carrier protein of the enzyme until itis liberated by the final enzymatic pocket on the enzyme, the intrinsicthioesterase. This thioesterase is the sole serine hydrolase within FAS,and is targeted by FP-TAMRA.

FAS is known to be up-regulated in a wide range of tumors, and itsfunction has been strongly linked to tumor cell proliferation, making itan attractive therapeutic target for cancer. We capitalized on the factthat FP-TAMRA reacts with the active site of all of the serinehydrolases visualized in FIG. 1 to a selectively identify an inhibitorof the FAS thioesterase domain.

Three beta-lactones, all derivatives of natural products, were testedfor the ability to block activity-based labeling of FAS. Interestingly,all three compounds had the ability to inhibit the thioesterase of FAS,but only tetrahydrolipstatin selectively inhibited FAS.Tetrahydroliptstatin, also known as orlistat, is a drug that has alreadybeen approved for weight management in obese patients. Interestinglyhowever, the effectiveness of orlistat in this indication is connectedto its ability to inhibit pancreatic lipase in the gastrointestinaltract, thereby preventing uptake of dietary fat. Such inhibition occursfollowing nucleophilic attack by the active site serine on the carbonylcarbon of the lactone ring. The reaction yields a covalent adductbetween enzyme and inhibitor (Hadvary et al. “The lipase inhibitortetrahydrolipstatin binds covalently to the putative active site serineof pancreatic lipase.” J. Biol. Chem. 266:2021-7, 1991). The inhibitionof FAS by orlistat has never been reported, and is not believed to berelevant to its mode of action in weight loss.

Studies were performed to determine the effect of orlistat on tumorcells. As a first step, we measured the ability to inhibit the activityof FAS in whole cells. PC-3 cells treated with a concentration range oforlistat for one hour were incubated with a membrane-permeableactivity-based probe, FP-Bodipy (2 μM), for an additional hour, and thelabeled cells were harvested and visualized as described above. Aconcentration-dependent inhibition of labeling by FP-Bodipy was evident(FIG. 2; top panel), indicating enzyme inhibition. These effects wereindependent of the abundance of FAS, which was measured from the sametreated samples by Western blot (FIG. 2; bottom panel). The effects oforlistat on cellular fatty acid synthesis were measured by assessing theincorporation of [¹⁴C]-acetate into fatty acids. A level of orlistatcapable of inhibiting about 90% of the activity-based labeling of FASreduced total cellular fatty acid synthesis by approximately 70%.

The effects of orlistat were not limited to inhibition of fatty acidsynthesis. This effect apparently has dramatic downstream consequences,because orlistat induced a pronounced apoptotic response in the DU-145and PC3 cells, which are the two more differentiated prostate lines. Asmall apoptotic response was observed in the LNCap cells, which are lessdifferentiated and which retain androgen responsiveness. Orlistat has noapoptotic effect on normal prostate epithelial (PrEC) cells, nor on aseries of normal human fibroblasts.

Methods

Profiling serine hydrolase activity in prostate cell lines. The LNCaP,DU-145 and PC-3 cell lines (ATCC, Rockville, Md.) were maintained inRPMI 1640 (Irvine Scientific, Santa Ana, Calif.) supplemented with 10%fetal bovine serum at 37° C. in 5% CO₂. The PrEC cell line (Clonetics,Walkerville, Md.) was maintained according to the suppliersinstructions. Each cell line was maintained in 150-mm tissue culturedishes. To generated protein lysates, cells were washed with ice-coldphosphate buffered saline (PBS) and harvested by scraping with a celllifter into cold PBS. Cells were collected by centrifugation and thepellets were resuspended in 50 mM Tris-Cl, pH 8.0. Lysis wasaccomplished by sonication and Dounce-homogenization as describedpreviously (Liu et al., supra; and Kidd et al. “Profiling SerineHydrolase Activities in Complex Proteomes.” Biochemistry 40:4005-4015,2001). The soluble and insoluble cell fractions were separated byultracentrifugation for one hour at 64,000 rpm at 4° C. Proteinconcentrations of the soluble fraction was determined by BCA assay(Pierce, Rockford, Ill.) versus a standard concentration of bovine serumalbumin (BSA).

The serine hydrolase activity profiles of the prostate cell lines weremeasured using the fluorophosphonate probe fp-PEG-Tamra using methodsdescribed previously (Liu et al., supra; and Kidd et al., supra).Briefly, 40 μl of a 1 mg/ml solution of the soluble fractions of eachcell lines were treated with 2 μM fp-PEG-Tamra for one hour at ambienttemperature. The labeling reactions were stopped by the addition ofLaemmli buffer followed by boiling for 5 minutes. As a control fornon-specific reaction of the probe, a duplicate sample was boiled forten minutes prior to labeling with fp-PEG-Tamra to denature allenzymatic activity. The labeled samples were resolved by 10% SDS-PAGEand visualized by scanning with a Hitachi flatbed scanner at 605 nm.

Alternatively, serine hydrolase activity in whole cells was measuredwith fp-Bodipy-Fl. Cells were plated in 24-well plates and the probe wasadded to a final concentration of 2 μM and labeled for one hour. Thecells were lysed by the addition of Laemmli sample buffer followed byboiling. The labeled samples were resolved by 10% SDS-PAGE andvisualized by scanning with a Hitachi flatbed scanner at 505 nm.

Inhibition of serine hydrolase activity with beta-lactone compounds.Ebelactone A and B stocks were made in DMSO. Orlistat was made in EtOH.Cell lysate were generated at 1 mg/ml as described above. Samples (40μg) were incubated with inhibitors for twenty minutes prior to additionof fp-PEG-TAMRA. The final concentration of DMSO or EtOH in eachreaction was 10%. The labeling reactions were stopped by the addition ofLaemmli sample buffer and samples were resolved on 10% SDS-PAGE andvisualized as described above.

Purification of serine hydrolases by avidin-biotin affinitychromatography. Serine hydrolases were purified from 2.5 mg of solublecell lysates by avidin-biotin affinity chromatography (Liu et al.,supra; and Kidd et al., supra). The lysates were pre-treated withavidin-agarose to remove non-specific binders. Lysates were labeled withfp-PEG-biotin (5 μM) for one hour at room temperature. Protein wasseparated from unincorporated fp-PEG-biotin by passage over a Nap 25column. Protein containing fractions were pooled and SDS was added to aconcentration of 0.5% and boiled for ten minutes. After boiling thesamples were diluted with 50 mM Tris, pH 7.5 and 150 mM NaCl.Avidin-agarose was added to the solution for a one-hour incubation atroom temperature. The agarose beads were pelleted by centrifugation andwashed eight times with 50 mM Tris, ph 7.5, 150 mM NaCl and 1% Tween 20.Labeled protein was eluted by the addition of Laemmli buffer containing1% SDS and boiling for ten minutes. Protein was resolved by 10% SDS-PAGEand detected by silver staining. Specific bands were extracted andsubjected to in-gel trypsin digests and MALDI-TOF analysis as describedpreviously (Landry et al. “A Method for Application of Samples toMatrix-Assisted Laser Desorption Ionization Time-of-Flight Targets ThatEnhances Peptide Detection.” Anal. Biochem. 279:1-8, 2000; Harvey et al.“Insights into a plasma membrane signature.” Physiol. Genomics.5:129-136, 2001).

Detection of fatty acid synthase by Western blot. PC-3 cells (5×10⁴)were seeded in 24 well plates. Following treatment with orlistat, cellswere collected, suspended in Laemmli sample buffer and boiled. Proteinwas resolved by 10% SDS-PAGE and transferred to nitrocellulose. Themembrane was blocked with non-fat milk and probed with an anti-FASmonoclonal antibody (mAb) (Pharmingen, San Diego, Calif.) followed byhorseradish peroxidase (HRP)-labeled rabbit anti-mouse IgG (BioRad,Hercules, Calif.) and chemiluminescence detection with Western LightingChemiluminescence Reagent (Perkin-Elmer, Boston, Mass.).

Inhibition of fatty acid synthesis by orlistat. Fatty acid synthesis incells was measured by [¹⁴C]-acetate incorporation (Kuhajda et al. “Fattyacid synthesis: a potential selective target for antineoplastictherapy.” Proc. Nat. Acad. Sci. U.S.A. 91:6379-83, 1994; Pizer et al.“Pharmacological inhibitors of mammalian fatty acid synthase suppressDNA replication and induce apoptosis in tumor cell lines.” Cancer Res.58:4611-5, 1998). Cells were seeded at density of 2.5×10⁴ cells/well in24-well plates. Prior to the addition of orlistat, the wells were washedtwice with PBS. Serum-free RPMI containing 300 μg/ml BSA andinsulin/transferrin/selenium supplement was added to the wells, with orwithout orlistat. The cells were incubated for two hours prior to theaddition of 1 μCi of [¹⁴C]-acetate to label newly synthesized fattyacids. After two hours, the labeling medium was removed and the cellswere washed with PBS/EDTA and trypsinized. The cells pellets were washedtwice more with PBS and fatty acids were extracted by the addition of anequal mixture of chloroform-methanol for 30 minutes. The extractedmaterial was dried under a stream of N₂ gas and extracted further withwater-saturated butanol. The butanol was removed by drying under astream of N₂ gas and labeled fatty acids were detected by scintillationcounting.

Detection of orlistat-induced apoptosis by annexin V labeling. Threeprostate cancer cell lines (LNCaP, DU-145 and PC-3) and humanfibroblasts (HF) were seeded in 35 mm plates. The cells were washedtwice with PBS and serum-free medium supplemented with 300 μg/ml BSA(Sigma, St. Louis, Mo.) and insulin/transferrin/selenium cocktail (LifeTechnologies, Rockville, Md.) containing various concentrations oforlistat was added. At various time points, the cells were harvested bytrypsinization and washed twice with PBS. Cells were suspended at 1×10⁶cells/ml in annexin V incubation buffer (BioVision, Inc., Mountain View,Calif.) and treated with annexin V-FITC and propidium iodide. Apoptoticcells were quantified by fluorescence-activated cell sorting (FACS)analysis.

Example II Orlistat Inhibits FAS Activity and Induces Cell Cycle Arrestand Apoptosis in Mammary Carcinoma Cells

This study focused on the identification of serine hydrolases active inmammary carcinoma. Activity-based protein profiling was combined with ascreen for small molecule antagonists to gain insight into the functionof these hydrolases. One of the prominent serine hydrolases, fatty acidsynthase, was found to be inhibited by tetrahydrolipstatin, a drugcommonly referred to as orlistat. Surprisingly, in mammary carcinomacells, orlistat elicits cell cycle arrest at the G₁/S boundary. In moredifferentiated tumor cells, apoptosis soon follows. These experimentsshow the relevance of fatty acid synthase as a therapeutic target andlink fatty acid synthesis to control of common cell cycle checkpoints.The study also reveals an unappreciated anti-tumor activity fororlistat, a drug approved for weight management in obesity.

Methods

Profiling Serine Hydrolase Activity in Mammary Epithelial Cells.

Primary human mammary endothelial cells (HMECs) and MCF-7, MDA-MB-231,and MDA-MB-435 cell lines (ATCC, Rockville, Md.) were maintained in RPMI1640 (Irvine Scientific, Santa Ana, Calif.) supplemented with 10% fetalbovine serum at 37° C. in 5% CO₂. Each cell line was maintained in150-mm tissue culture dishes. To generated protein lysates, cells werewashed with ice-cold phosphate buffered saline (PBS) and harvested byscraping with a cell lifter into cold PBS. Cells were collected bycentrifugation and the pellets were resuspended in 50 mM Tris-Cl, pH8.0. Lysis was accomplished by sonication and Dounce homogenization asdescribed previously (Liu et al. “Activity-based protein profiling: theserine hydrolase.” Proc. Natl. Acad. Sci. U.S.A 96:14694-9, 1999; Kiddet al. “Profiling Serine Hydrolase Activities in Complex Proteomes.”Biochemistry 40:4005-4015, 2001). The soluble and insoluble cellfractions were separated by ultracentrifugation for one hour at 64,000rpm at 4° C. Protein concentrations of the soluble fraction wasdetermined by BCA assay (Pierce, Rockford, Ill.) versus a standardconcentration of bovine serum albumin (BSA).

The serine hydrolase activity profiles of the prostate cell lines weremeasured using the fluorophosphonate probe fp-PEG-TAMRA using methodsdescribed previously (Liu et al., supra; Kidd et al., supra). Briefly,40 μl of a 1 mg/ml solution of the soluble fractions of each cell lineswere treated with 2 μM fp-PEG-TAMRA for one hour at ambient temperature.The labeling reactions were stopped by the addition of Laemmli bufferfollowed by boiling for 5 minutes. As a control for non-specificreaction of the probe, a duplicate sample was boiled for ten minutesprior to labeling with fp-PEG-TAMRA to denature all enzymatic activity.The labeled samples were resolved by 10% SDS-PAGE and visualized byscanning with a Hitachi flatbed scanner at 605 nm.

Alternatively, serine hydrolase activity in whole cells was measuredwith fp-Bodipy-Fl. Cells were plated in 24-well plates and the probe wasadded to a final concentration of 2 μM and labeled for one hour. Thecells were lysed by the addition of Laemmli sample buffer followed byboiling. The labeled samples were resolved by 10% SDS-PAGE andvisualized by scanning with a Hitachi flatbed scanner at 605 nm.

Inhibition of Serine Hydrolase Activity with Beta-Lactone Compounds.

Ebelactone A and B stocks were made in DMSO. Orlistat was made in EtOH.Cell lysate were generated at 1 mg/ml as described above. Samples (40μg) were incubated with inhibitors for twenty minutes prior to additionof fp-PEG-TAMRA. The final concentration of DMSO or EtOH in eachreaction was 10%. The labeling reactions were stopped by the addition ofLaemmli sample buffer and samples were resolved on 10% SDS-PAGE andvisualized.

Purification of Serine Hydrolases by Avidin-Biotin AffinityChromatography.

Serine hydrolases were purified from 2.5 mg of soluble cell lysates byavidin-biotin affinity chromatography (Liu et al., supra; Kidd et al.,supra). The lysates were pre-treated with avidin-agarose to removenon-specific binders. Lysates were labeled with fp-PEG-biotin (5 μM) forone hour at room temperature. Protein was separated from unincorporatedfp-PEG-biotin by passage over a Nap 25 column. Protein containingfractions were pooled and SDS was added to a concentration of 0.5% andboiled for ten minutes. After boiling the samples were diluted with 50mM Tris, pH 7.5 and 150 mM NaCl. Avidin-agarose was added to thesolution for a one-hour incubation at room temperature. The agarosebeads were pelleted by centrifugation and washed eight times with 50 mMTris, ph 7.5, 150 mM NaCl and 1% Tween 20. Labeled protein was eluted bythe addition of Laemmli buffer containing 1% SDS and boiling for tenminutes. Protein was resolved by 10% SDS-PAGE and detected by silverstaining. Specific bands were extracted and subjected to in-gel trypsindigests and MALDI-TOF analysis as described previously (Landry et al. “AMethod for Application of Samples to Matrix-Assisted Laser DesorptionIonization Time-of-Flight Targets That Enhances Peptide Detection.”Anal. Biochem. 279:1-8, 2000; Harvey et al. “Insights into a plasmamembrane signature.” Physiol. Genomics. 5:129-136, 2001).

Detection of Fatty Acid Synthase by Western blot.

MDA-MB-435 cells (5×10⁴) were seeded in 24 well plates. Followingtreatment with orlistat, cells were collected, suspended in Laemmlisample buffer and boiled. Protein was resolved by 10% SDS-PAGE andtransferred to nitrocellulose. The membrane was blocked with non-fatmilk and probed with an anti-FAS mAb (Pharmingen, San Diego, Calif.)followed by HRP-labeled rabbit anti-mouse IgG (BioRad, Hercules, Calif.)and chemiluminescence detection with Western Lighting ChemiluminescenceReagent (Perkin-Elmer, Boston, Mass.).

Inhibition of Fatty Acid Synthesis by Orlistat.

Fatty acid synthesis in cells was measured by [¹⁴C]-acetateincorporation (Kuhajda et al. “Fatty acid synthesis: a potentialselective target for antineoplastic therapy.” Proc. Nat. Acad. Sci.U.S.A. 91:6379-83, 1994; Pizer et al. “Pharmacological inhibitors ofmammalian fatty acid synthase suppress DNA replication and induceapoptosis in tumor cell lines.” Cancer Res. 58:4611-5, 1998). Cells wereseeded at density of 2.5×10⁴ cells/well in 24-well plates. Prior to theaddition of orlistat, the wells were washed twice with PBS. Serum-freeRPMI containing 300 μg/ml BSA and insulin/transferrin/seleniumsupplement was added to the wells, with or without orlistat. The cellswere incubated for two hours prior to the addition of 1 μCi of[¹⁴C]-acetate to label newly synthesized fatty acids. After two hours,the labeling medium was removed and the cells were washed with PBS/EDTAand trypsinized. The cells pellets were washed twice more with PBS andfatty acids were extracted by the addition of an equal mixture ofchloroform-methanol for 30 minutes. The extracted material was driedunder a stream of N₂ gas and extracted further with water-saturatedbutanol. The butanol was removed by drying under a stream of N₂ gas andlabeled fatty acids were detected by scintillation counting.

Detection of Orlistat-Induced Apoptosis.

Three breast cancer cell lines (MDA-MB-435, MDA-MB-231, and MCF-7) andhuman mammary epithelial cells (HMEC) were seeded in 96-well plates at1×10⁴ cells per well. The cells were washed twice with PBS and thenincubated in serum free medium supplemented with 300 μg/ml BSA (Sigma)and insulin/transferrin/selenium cocktail (Life Technologies, Rockville,Md.) containing the indicated concentrations of orlistat. Aftertwenty-four hours, the media was removed and the Cell Death DetectionELISA^(plus) kit (Roche, Indianapolis, Ind.) was used to measure DNAfragmentation. The raw data were transformed to % cell death based onpositive control standards.

Results

The Serine Hydrolase Profile of Mammary Carcinoma.

An activity-based probe was used to define the serine hydrolase profileof breast cancer cell lines. The probe is comprised of afluorophosphonate warhead linked to the TAMRA fluorophore (FP-TAMRA)(Patricelli et al. “Direct visualization of serine hydrolase activitiesin complex proteomes using fluorescent active site-directed probes.”Proteomics 1:1067-1071, 2001). Primary cultures of normal mammaryepithelial cells (HMEC) were compared to three breast cancer cell lines,MCF-7, MDA-MB-231 and MDA-MB-435. These three lines represent a spectrumof phenotypes (Kurebayashi et al. “Quantitative demonstration ofspontaneous metastasis of MCF-7 human breast cancer cells co-transfectedwith fibroblast growth factor 4 and LacZ.” Cancer Res. 53:2178-2187,1993; Shafie et al. “Formation of metastasis by human breast carcinomacells (MCF-7) in nude mice.” Cancer Lett. 11:81-87, 1980; and Price etal. “Tumorigenicity and metastasis of human breast carcinoma cell linesin nude mice.” Cancer Res. 50:717-721, 1990). MCF-7 cells are estrogenresponsive and non-invasive. The other two lines have lost estrogencontrol and are invasive in animals. Lysates from each cell type werereacted with FP-TAMRA and then resolved on SDS-PAGE (FIG. 3). In eachcase approximately fifteen different active hydrolases are visualized asfluorescent bands on SDS gels. The pattern of serine hydrolaseexpression is generally similar among the cell lines.

Screening for Serine Hydrolase Inhibitors.

We capitalized on the fact that FP-TAMRA reacts at the active site ofall of the serine hydrolases visualized in FIG. 3, by performing asimultaneous screen for antagonists of all of these enzymes. Threebeta-lactones were tested for the ability to block labeling of themammary serine hydrolases by FP-TAMRA: ebelactones A and B, andorlistat.

Labeling of serine hydrolases in the cell lysates with FP-TAMRA waschallenged with each of the beta-lactones. Inhibition experiments wereperformed under pre-steady state conditions, so that relative IC₅₀values could be compared. All three compounds blocked labeling ofhydrolases by FP-TAMRA (FIG. 4), indicating inhibition at the activesite serine. Interestingly though, each compound exhibited a differentspectrum of inhibition. For example, ebelactone A was a potent inhibitorof the hydrolase expressed at 28 kDa, but ebelactone B had little effecton this enzyme. Conversely, ebelactone B abolished labeling of ahydrolase migrating at 31 kDa, but the same concentration of ebelactoneA was far less effective. Tetrahydrolipstatin is selective for thehydrolase at 270 kDa that is expressed in breast cancer cell lines.

To identify the hydrolases that are inhibited by each beta-lactone, aslightly different labeling strategy was used. Cell lysates were reactedwith a biotinylated derivative of fluorophosphonate (Liu et al., supra).Then, the tagged hydrolases were subjected to affinity purification onavidin-agarose columns. Bands in the eluate corresponding to hydrolaseshit by the beta-lactones were excised and subjected to peptide massfingerprinting by MALDI-TOF mass spectrometry. In some instances theidentification was confirmed by MS/MS sequencing. The 270 kDa band thatis inhibited exclusively by orlistat was found to be fatty acid synthase(FAS). The identity of the protein was confirmed by immunoprecipitatingthe complex between FP-TAMRA and FAS with an anti-FAS monoclonalantibody.

Orlistat Inhibits FAS in Whole Cells and Blocks Cellular Fatty AcidSynthesis.

Studies were performed to determine whether orlistat could block thebiological function of FAS in whole cells. MDA-MB-435 cells treated witha range of orlistat were probed with a membrane-permeable activityprobe, FP-Bodipy. A concentration-dependent inhibition of the labelingof cellular FAS by orlistat was evident. These effects were independentof the abundance of FAS, which was measured from the same treatedsamples by Western blot. We measured the effects of orlistat on cellularfatty acid synthesis. MDA-MB-435 cells were fed [¹⁴C]-acetate as aprecursor, and treated with orlistat. At 100 μM orlistat theincorporation of [¹⁴C]-acetate into cellular fatty acids was reduced byapproximately 70%. This observation is taken to indicate that thebiological activity of FAS in tumor cells is drastically reduced byorlistat.

Induction of Tumor Cell Apoptosis by Orlistat.

The effect of orlistat on apoptosis in the MDA-MB-435, MDA-MB-231 orMCF-7 mammary carcinoma cell lines, or in primary cultures of normalmammary epithelial cells or fibroblasts (HF cells; ATCC, Rockville,Md.), was measured using DNA fragmentation as an indicator. Cells(1×10⁴/well) were treated with orlistat in defined medium for 48 hours.Cells were lysed and DNA fragmentation was measured by ELISA andnormalized to apoptosis induced by camptothecin. In each case triplicatemeasurements were made with a standard deviation of less then 10%.

Interestingly, orlistat induced an apoptotic response in all three tumorlines, without effect on normal HMECs (FIG. 5) nor on fibroblasts. Theapoptotic response was most pronounced in the more differentiated tumorlines, MDA-MB435 and MDA-MB-231. Orlistat had only a moderate effect onthe MCF-7 cells. This analysis was extended by comparing the effect oforlistat on MDA-MB-435 cells and the HMECs across a range of orlistat.Half-maximal response in the MDA-MB-435 cells was observed at about 4 uMorlistat. No effect was evident in the HMECs.

Orlistat Induces G₁/S cell Cycle Arrest.

To determine if the apoptotic effects of orlistat are associated with acell cycle checkpoint, the effect of orlistat on cell cycle progressionof the MDA-MB-435 cells was measured by staining cellular DNA withpropidium iodide, and then assessing DNA content by flow cytometry.Treatment with orlistat caused a pronounced increase in the percentageof cells present in G₁, and a corresponding decrease in the percent ofcells in S phase (FIG. 6: G₁=dark bars, S=open bars, and G₂/M=greybars). These observations link the inhibition of FAS by orlistat to theRb axis that controls G₁/S progression.

Example III Orlistat Inhibits Endothelial Cell Proliferation and FASActivity

The experiments described below provide the first demonstration thathuman endothelial cells express FAS, and show that inhibition of FASwith orlistat induces a GI cell cycle block and inhibits endothelialproliferation.

Results

Orlistat Inhibits the Serine Hydrolase Activity of HUVEC Fatty AcidSynthase.

An activity-based probe, composed of a fluorophosphonate reactive grouplinked to the Tamra fluorophore (fp-TAMRA), was used to define theserine hydrolase profile of human umbilical vein endothelial cells(HUVEC). A cell lysate was allowed to react with fp-TAMRA and theproteins resolved by SDS-PAGE. Approximately 19 different hydrolaseswere detected as fluorescent bands on the gel. An intense bandidentified as fatty acid synthase (FAS) was evident close to the 220 kDamolecular weight marker.

Orlistat was assessed as an inhibitor of endothelial serine hydrolasesby pre-incubating cell lysates with orlistat in a range ofconcentrations. FAS was the only hydrolase affected, showing a selectivereduction in subsequent binding of fp-TAMRA. Orlistat inhibited FASactivity by a maximum of 80%, and orlistat concentrations of 5-10 uMwere required to reach this level of inhibition. The half maximal effectwas accomplished by 660 nM orlistat.

Orlistat Inhibits Proliferation of HUVECs.

The effect of orlistat on HUVEC proliferation induced by severalmitogenic stimuli was assessed by BrdU incorporation. Orlistat andmitogen were added simultaneously to serum-starved cells, and BrdUincluded during the period equivalent to the second round of the cellcycle after mitogen addition. Orlistat inhibited proliferation inducedby complete endothelial medium (containing a undefined mixture ofmitogens), by bFGF and by VEGF. The minimal amount of proliferationoccurring in basal medium supplemented with 0.2% fetal calf serum (FCS)only was also inhibited. Maximum inhibition was almost 90% for cellsstimulated with complete medium or bFGF, but nearer to 80% when VEGF wasused, and was achieved at orlistat concentrations of 10 uM and above.Orlistat concentrations required for a half maximal effect were 4 uM, 1uM and 1.25 uM respectively.

Orlistat Induces G1 Cell Cycle Arrest.

Progression of HUVEC through the cell cycle was monitored by flowcytometric assessment of DNA content. After 24 h serum starvation, fewcells were undergoing cell division; the distribution of cells betweenG1, S and G2/M phases was 67%, 6% and 27%, respectively. In the absenceof orlistat, cells re-exposed to Endothelial Growth Medium entered Sphase 12 h later, with DNA synthesis reaching a population maximum at 16h. The percentage of cells in G2/M peaked 4 h later, at 20 h, afterwhich the cells rapidly re-entered S phase. In the presence of 10 uMolistat, entry into the first round of S phase was partially blocked,with a corresponding increase in the percentage of cells remaining inG1, and decrease in cells progressing on to G2/M phase. The second Sphase peak was completely inhibited by orlistat, with the cellsremaining in G1. These observations indicate that orlistat induces a G1cell cycle block in HUVECs.

Methods

Effect of Orlistat on FAS Activity in HUVEC Lysates.

HUVEC lysates (40 ug) were preincubated for 30 min with orlistat atconcentrations ranging from 10-0 uM, followed by a one hour incubationwith fp-TAMRA to tag any remaining active sites. Non-specific labelingwith the probe was determined by boiling samples prior to the additionof fp-Tamra. Reactions were stopped by addition of SDS loading buffer,followed by boiling. Proteins (30 ug/lane) were resolved by SDS-PAGEusing a 10% gel, and visualized at 605 nm using a Hitachi flat bed gelscanner. The density of the band corresponding to FAS was measured usingImage Analysis v2.0 software (Hitachi Genetic Systems) and the effect oforlistat on FAS activity was expressed as a percentage of the bandintensity in the untreated lysate.

Effect of Orlistat on Endothelial Cell Proliferation.

Cell proliferation was measured by incorporation of BrdU. HUVEC wereseeded in 96 well plates at a density of 2000 cells/well and cultured inEndothelial Growth medium (EGM; Clonetics, Walkerville, Md.) for 24 h at37° C. Cells were serum-starved for further 24 h in Endothelial BasalMedium (EBM; Clonetics, Walkerville, Md.)+0.2% FCS. Medium was replacedwith orlistat (40-0 uM) diluted in (A) EGM, (B) EBM+2% FCS+bFGF (5ng/ml), (C) EBM+2% FCS+VEGF (20 ng/ml) or (D) EBM+0.2% FCS. Cells wereincubated 48 h at 37 deg C., with BrdU present during the final 24 h.Medium was removed, the cell monolayers fixed, and BrdU incorporationmeasured by ELISA. Proliferation was expressed as a percentage of thatmeasured in the absence of orlistat.

Effect of Orlistat on HUVEC Passage Through the Cell Cycle.

HUVEC were seeded into 6-well plates at a density of 74,000 cells/welland cultured in EGM for 24 h at 37 deg C. Cells were serum-starved forfurther 24 h in EBM+0.2% FCS. Medium was replaced with EGM+/−10 uMorlistat, and cells cultured for up to 38 h more. Untreated andorlistat-treated cells were sampled at 4 h intervals. The DNA content ofcells was assessed by binding of propidium iodide, and percentage ofcells in (A) G1 phase, (B) S phase and (C) G2/M phase at each time pointwas calculated.

Example IV Sequential in situ Ketene Generation, Dimerization andHydrogenation for the Catalytic, Asymmetric Synthesis of β-Lactones

The current discovery that beta-lactones inhibit serine hydrolaseactivity prompted development of the following novel process forsynthesizing optically active beta-lactones from achiral startingmaterial.

Results

The development of a concise, asymmetric route to psuedosymmetric3,4-dialkyl-cis-beta-lactones, analogous to the FDA approvedanti-obesity agent tetrahydrolipstatin is reported in this example. Theprocess is based a two-step process of ketene dimerization/hydrogenationfrom acid chlorides, followed by subsequent alpha-epimerization andalpha-alkylation or acylation leading to beta-lactones bearingquaternary carbons. These beta-lactones displayed antagonistic activity(apparent Ki's in the .micro.M range) in competition with a fluorogenicsubstrate toward a recombinant form of the thioesterase domain of fattyacid synthase, which has great potential as a new therapeutic target forcancer.

A Novel Two-Step Synthesis Method for Obtaining Asymmetric Beta-Lactonesfrom Acid Chloride.

According to the present invention there is provided a process ofpreparing achiral beta-lactones comprising the steps of: forming aketene dimer from an acid chloride and hydrogenating the ketene dimer togenerate a cis-beta-lactone. The invention process is generallyrepresented by Scheme I

Step 1: Formation and Isolation of Ketene Dimers.

In forming a ketene dimer by the current method, an acid chloride havingthe formula VI is used as a starting material. The acid chloride has theformula VII:

wherein R can be a hydrogen, an alkyl group, a cycloalkyl group, aheterocycloalkyl group, an alkoxy group, a hydroxyalkyl group, ahalogenated alkyl group, an alkoxyalkyl group, an alkenyl group, analkynyl group, an aryl group, a heteroaryl group, an aralkyl group, ahydroxy group, an amine group, an amide, an ester, a carbonate group, acarboxylic acid, an aldehyde, a keto group, an ether group, a halide, aurethane group, a silyl group, a sulfo-oxo group.

Previous large scale synthesis and purification of racemic ketene dimersbearing long alkyl chains (>13 carbons) relied on acidic extraction toremove alkyl ammoniums salts followed by vacuum distillation. Becausemany functionalities are acid sensitive, the current synthesis methodpreferably utilizes silica gel chromatography. Ketene dimers werepurified by silica gel flash chromatography although the yields weredecreased relative to direct transformation. The yield of ketene dimerwas improved slightly by use of doubly distilled acid chloride (78% vs62% yield, Table 1, entry 5).

TABLE 1

entry catalyst time (h) % yield^(d) % ee^(c) 1 QND (9) 24 58 98 2 O-TBSQND (10) 24 54 ND 3 O-TMS QND (10) 24 55 ND 4 O-TMS QUIN (12)  6 62 ND 5O-TMS QUIN (12)  6  78^(b) 96 ^(a)Yields refer to isolated, purifieddimer. ^(b)Freshly, doubly distilled acid chloride was used. ^(c)ND =not determined. QND means_quinidine; O-TBS QND means O-t-butyl dimethylsilyl quinidine; O-TMS QND means_O-trimethylsilyl quinidine; O-TMS QUINmeans O-trimethylsilyl quinine.

One ketene dimer, referred to as 3a in Table 2 below, possessedsufficient stability to determine its etantiomeric purity by gaschromatography using cyclodextrin bis-OTBS as chiral stationary phase.Both enantiomers of ketene dimer 3a were obtained in high optical purityusing either QND (>98% ee) or O-TMS QUIN (>96% ee). The enantiopurity ofother dimers were determined following hydrogenation to thecis-beta-lactones (vide infra). We found that silylated alkaloids weresuperior catalysts in terms of reaction efficiency for the ketenedimerization step as compared to acetylated derivatives (formed in situ)as has been previously reported by Calter. (See, M. A. Calter, J. Org.Chem. 1996, 61, 8006; M. A. Calter, R. K. Orr, W. Song, Org. Lett. 2003,5, 4745; and R. K. Orr, M. A. Calter Tetrahedron 2003, 59, 3545. )

Step 2: Hydrogenation of Ketene Dimers.

Prior hydrogenation studies of diketenes have primarily focused on theparent diketene, 4-methylene-2-oxetanone, in both racemic and asymmetricfashion, as a means to obtain the corresponding3,4-dimethyl-2-oxetanone, which is a commodity chemical utilized on tonscale for polymer applications. Several catalysts have been utilized forhydrogenation of enol ethers, however for simplicity and practicalitypalladium on carbon is initially used in this example with racemicketene dimer 2a (entry 1, Table 2).

At the outset, we expected high facial selectivity for the hydrogenationdue to the proximity of the alkene to the alpha-stereogenic carbon at C3of the beta-lactone. A high degree of diastereoselectivity was observedduring epoxidations of many of these ketene dimers. However,hydrogenation of dimer 2a employing 5 mol % Pd/C (5 wt %) resulted inhigh yield but low diastereoselectivity providing a mixture of cis- andtrans-beta-lactones 3a (4:1 favoring cis) after a 24 h reaction time(entry 1, Table 2). To reduce the activity of the catalyst, 100 mol %triethylamine relative to Pd catalyst was added. The addition oftriethylamine also improved the diasteroselectivity to 17:1 with thesame reaction time (entry 2, Table 3). However, repeating theseconditions with optically active dimer 2a (98% ee) indicated thatracemization was occurring under these conditions necessarily at thedimer stage providing cis-beta-lactone 3a with reduced enantiopurity(71% ee). Shortening the reaction time using 5 mol % catalyst withoutadded Et3N also gave high diastereoselectivity. Taken together, theseresults suggest that either long reaction times or the absence ofcatalyst poison leads to erosion in diastereoselectivity. Optimalconditions that prevented racemization, maintained highdiastereoselectivity, and reduced reaction time were eventually realizedby decreasing the amount of Pd/C to 1 mol % and reducing the reactiontime to 30 min under 30 psi of H.sub.2 pressure. Under these conditions,no racemization or epimerization was observed for either of the ketenedimer or major diastereomer isolated, cis-beta-lactone 3a. The lattercould be isolated in 90% yield and 96% ee and >19:1 diastereomeric ratio(Table 3, entry 4) as determined by coupling constant analysis(JHa,Hb=6.3 Hz).

TABLE 2 Catalytic asymmetric beta-lactone synthesis via a sequential,two-step ketene dimerization/hydrogenation sequence. % % % yield yieldee entry R ketene dimer (2) (2)^(b) beta-lactone (3) (3)^(c) (3)^(d) 1n-butyl

75

90 96 2 cyclopentyl

54

89 94 3 cyclohexyl

55

89 90 4 benzyl

48

85 96 5 CH₂CO₂Me

 60^(e)

94 92 6 11-methoxynonyl

 62^(f)

94 ND ^(a)All reactions were carried out at 0.1M (final concentration)with freshly distilled acid chloride. ^(b)Refers to isolated, purifiedyields. ^(c)Enantiomeric excess was determined by chiral GC analysis.^(d)Absolute configuration of the major enantiomer is depicted.^(e)Reaction time was 3 h at 0° C. ^(f)Reaction time was 3.5 h with 5mol % catalyst 7.

TABLE 3 Effect of catalyst loading, base, and reaction time ondiastereoselectivity and enantioselectivity.^(a) mol % mol % time %entry Pd/C Et₃N (h) yield dr^(b) 1 5 0 24 88  4/1 2 5 100 24 92 17/1 3 50 0.5 89   17:1 4 1 0 0.5 90 >19:1^(c) ^(a)Reactions were conducted at0.1 M in CH2Cl2. ^(b)Ratios estimated by 1H NMR (500 MHz) integration ofHa and Hb in crude reaction mixtures. ^(c)Enantiomeric excess determinedby GC analysis to be 98% ee.

In this example it is shown that the hydrogenation of a series of ketenedimers 2a-f (Table 2) results in consistently high yields of thecorresponding beta-lactones 3a-f (Table 2) with high enantiomericpurities (Table 3). Enantiomer ratios were determined by chiral GCanalysis following hydrogenation. Beta-Lactone 3c was crystalline andthus x-ray analysis verified the cis stereochemistry obtained duringhydrogenation (FIG. 7). Compounds that have been synthesized by thismethod are shown in FIGS. 10, 11 and 12.

One-Pot Ketene Dimerization Sequence.

In an alternative embodiment of the novel two-step synthesis method, thereaction is carried out as a one-pot, two step ketenedimerization/hydrogenation process. (Scheme IV). In the example thereaction employs hexanoyl chloride to access the correspondingbeta-lactone. (entry 1, Table 2) Following ketene dimerization by themethod of Calter employing quinidine (FIG. 8) and simple filtration toremove amine hydrochloride salts, the reaction was transferred to ahydrogenation vessel and pressurized to 30 psi of H.sub.2. Thisprocedure provided only modest yields of beta-lactone 3a in this mannerdue to presumed degradation of the ketene dimers in the presence oftraces of dissolved quaternary ammonium salts, a process with precedentin the literature.

Despite careful filtration of the amine salts prior to hydrogenation,extensive degradation of the dimer was observed. More importantly, theenantiomeric purity of the ketene dimer was found to erode during thehydrogenation, (97% to 78% ee at 40% conversion) and thus the purity ofthe beta-lactone (97% to 74% ee). This method, therefore, is lessdesirable than the preferred embodiment for obtaining the desiredoptically enriched beta-lactones. The stereochemistry of beta-lactone 3awas assigned based on coupling constant analysis (Jcis ˜6 Hz, Jtrans4-4.5 Hz) and subsequently confirmed by x-ray analysis of beta-lactone3c (FIG. 7).

Reaction Analysis by Reaction View Spectroscopy.

The dimerization and related processes were monitored by in situ IR(Reaction View, Scientific Optical Solutions Ltd, Cathcart, Glasgow,Scotland) spectroscopy. The formation of ketene from hydrocinnamoylchloride (entry 4, Table 2) followed by addition of MeOH to form thecorresponding methyl ester was analyzed first. Interestingly, generationof ketene appears complete within a short time and persists until MeOHis added leading to a fast conversion to methyl cinnamate. Adimerization process leading to the phenyl bearing ketene dimer 3d wasstudied at 23 . deg.C. using quinuclidine hydrochloride as nucleophiliccatalyst and Hunig's base as stoichiometric. Base (FIGS. 9 a and 9 b).Addition of hydrocinnamyl chloride (1793 cm⁻¹) leads to a briefappearance of ketene (2118 cm⁻¹) with concomitant formation of ketenedimer 2d (1710 cm⁻¹). The disappearance of ketene and acid chlorideprior to complete formation of ketene dimer is suggestive of a very fastdimerization step or generation of another intermediate that isshort-lived. Indeed, dimerization is complete within 1 hour, as shown.

Beta-Lactones that are Structurally Similar to Orlistat can be Obtainedfrom Acid Chlorides in Three Steps.

The two-step synthesis method described directly above can subsequentlyinclude the steps of, epimerizing the cis-beta-lactone to produce thetrans-beta-lactone in a mix, or alkylating or acylating thecis-beta-lactone to form trisubstituted-beta-lactones. These subsequentprocesses are generally presented in Scheme II and Scheme III,respectively.

Epimerization of cis-beta-lactones.

As described above, the majority of naturally-occurring beta-lactonespossess trans-beta-lactone stereochemistry including Orlistat. Thus,this example describes conditions to epimerize to the thermodynamicallypreferred trans-beta-lactones. Interestingly, deprotonation leading tobeta-lactone enolates is possible since beta-elimination in thesesystems is a symmetry forbidden process. Competing Claisen condensationcan be precluded provided there is an alpha-substituent. Severalthermodynamic conditions to effect epimerization were studied and themost promising was that using a triethylamine/ammonium acetate buffersystem in dichloromethane. However, use of this buffer system only ledto from 10 to 15% upto ˜40% conversion diastereomeric ratio (dr) by 1HNMR analysis after prolonged stirring at 23 . deg.C. After someinvestigation, we found that deprotonation with lithiumhexamethyldisilazide under kinetic conditions followed by lowtemperature quenching with acetic acid provided a mixture of cis- andtrans-isomers in moderate yield which could be separated bychromatography (Scheme V).

Specific examples of epimerized beta-lactone compounds that have beensynthesized by this process are shown in FIG. 11. Moreover, synthesizedbeta-lactones closely resembling tetrahydrolipstatin and pancilicin Dare shown in FIG. 12.

Alkylation/acylation of cis-beta-lactones to form trisubstitutedbeta-lactones.

Cis-beta-lactone structures synthesized using the novel synthesis methodof the current invention can be further modified to produce diastereomercompounds.

For example, enolization with LDA followed by addition of variouselectrophiles allowed access to beta-lactones bearing alpha-quaternarycarbons. These reactions proceeded with high diastereoselectivities forbulky alkylating and acylating agents whereas smaller electrophiles suchas methyl iodide provided moderate diastereoselectivity (entry 1, Table4; dr, 4:1). Use of LiHMDS and NaHMDS led to similar yields andselectivities in the case of beta-lactones 8b and 8c, respectively.Compounds that have been synthesized by this method are shown in FIGS.11 and 12.

TABLE 4

Diastereoselective alkylations and acylations of cis-beta-lactone-4aleading to quaternary carbon bearing beta-lactones 8a-c. entry R cmpd.no. base % yield^(a) dr^(b) 1 CH₃ 8a LDA 80  4:1 2 Benzyl 8b LiHMDS 6119:1 3 CO₂Benzyl 8c NaHMDS 74 19:1 ^(a)Refers to isolated, purifiedyield. ^(b)Diastereomeric ratio was determined by integration (1H NMR500 MHz).

Methods

General Experimental Procedure for Dimerization as Described for (R,Z)-3-butyl-4-pentylideneoxetan-2-one (2a). To a flame dried 1 L roundbottom flask was added 764 mg (5 mol %, 1.926 mmol) TMS-quinine, 385 mLCH₂Cl₂ (0.1M) and 6.86 mL (1.0 equiv, 38.53 mmol) of Hunig's base undernitrogen atmosphere at 22° C. To this colorless solution, 5 mL (5.18 g,38.53 mmol) of freshly double-distilled, hexanoyl chloride was addedover 15 min via syringe. After 6 h the dark yellow solution wasconcentrated down to 100 mL (⅕ original volume) in vacuo and 250 mL ofpentanes was added to precipitate the ammonium salts. Filtration throughWhatmann filter paper (#1, qualitative grade), concentration in vacuoand purification by flash column chromatography on deactivated SiO₂ (10%H₂O) (2.5 cm×35.0 cm column, 15 cm pad) eluting with 0→20% Et₂O: hexanesgave 2.83 g (75%) of 2a as a colorless oil (96% ee, chiral GC analysis).R_(f)0.54 (15% Et₂O:hexanes); IR (thin film) 1865 cm⁻¹; ¹H NMR (300 MHz,CDCl₃) δ 4.70 (dt, J=6.3, 1.3 Hz, 1H), 3.94 (dt, J=1.0, 7.0 Hz, 1H),2.13 (app q, 2H), 1.75-1.83 (m, 2H), 1.28-1.53 (m, 8H), 0.88-0.96 (m,6H); ¹³C NMR (300 MHz, CDCl₃) δ 14.5, 14.6, 22.9, 23.1, 25.1, 28.0,29.2, 32.3, 54.45, 102.4, 146.4, 170.6; ESI LRMS Calcd. For C₁₂H₂₀O₂[M+Li]: 202; Found: 202.

Racemic ketene dimers were initially prepared in a similar manner usingHünig's base. Subsequently, 5 mol % quinuclidine hydrochloride with 1.0equiv of Hunig's base gave optimal results (reaction rate and yield)therefore this method was used for preparation of racemic ketene dimers.

Representative Procedure for Hydrogenation as Described for (3R,4S)-3-butyl-4-pentyloxetan-2-one (3a).

The purified n-butyl ketene dimer 2a, (5.1 mmol, 1.0 g) was dissolved in40 mL dichloromethane (0.1 M) and transferred to a Parr bomb apparatusunder a N.sub.2 atmosphere using an additional 10 mL dichloromethane aswash solvent. Pd/C (107 mg, 1 mol %, 5 wt %) was then added at one timeunder N.sub.2 atmosphere. The Parr bomb was then subjected to threeconsecutive evacuation-saturation cycles of hydrogen gas and thenpressurized to 30 psi hydrogen gas pressure. Hydrogenation with shaking(Parr shaker) was continued for 30 min at this pressure and then theheterogeneous slurry was vacuum filtered through a plug of Celite, andconcentrated yielding a colorless oil. Flash chromatography withgradient elution (5→15% diethyl ether/hexanes; 2.5×35.0×5 cm pad) gave3-butyl-4-pentyl-oxetan-2-one (3a, 897 mg, 90%) as a colorless oil (>96%ee, chiral GC): Rf 0.47 (15% Et2O:hexanes ); IR (thin film) 1824 cm−1;1H NMR (300 MHz, CDCl.sub.3) δ 4.54 (ddd, J=2.1, 3.6, 5.7 Hz, 1H), 3.59(ddd, J=4.5, 5.4, 8.4 Hz, 1H), 1.46-1.84(m,6H), 1.31-1.44 (m, 8H),0.88-0.94 (m, 6H); 13C NMR (500 MHz, CDCl₃) δ 173.1, 76.5, 53.3, 32.2,30.9, 30.5, 26.0, 24.4, 23.2, 23.2, 14.7, 14.5; ESI LRMS Calcd. forC12H22O2 [M+Li]: 205: Found:205.

The enantiomeric purity of ketene dimer 2a and β-lactone 3a wasdetermined to be >96% e.e by chiral GC analysis. Column Type: chiralbis-OTBS-cyclodextrin; retention time: tdimer 16.96 (major), tdimer17.16 (minor); tβ-lactone 26.23 (major), tβ-lactone 26.44 (minor).Conditions: make up flow: 25 mL/min; H2 flow: 30 mL/min; air flow: 300mL/min; injector: temperature: 200° C., pressure: 5 psi (hold time: 30min); oven: temperature gradient: 100- 140° C. (hold time: 30 min);detector temperature: 250° C.

(3R, 4R)-3-butyl-4-pentyloxetan-2-one (3a)

To a −78 ° C. solution of 100 mg (0.51 mmol) of 3a in 5 mL THF was added760 μL LiHMDS (1.5 equiv, 1.0 M in THF) and allowed to stir for 1 h.Tetramethylenediamine (TMEDA, 120 μL, 1.5 equiv, 0.76 mmol) was thenadded at −78° C. and allowed to stir for an additional 30 min afterwhich the solution was quenched with glacial acetic acid (130 μL, 3.0 18equiv, 2.27 mmol) and the mixture was warmed to 22° C. After extractionwith diethyl ether (2×6 mL), the combined organics were washed with 2 mLpH 7.0 buffer and 2 mL brine and then dried over Na2SO4. Concentrationin vacuo gave a colorless oil which upon purification by flashchromatography on SiO₂ (15% Et2O: hexanes) gave cis-3a and trans-3a (72mg, 72% yield) as a 1:1 mixture of diastereomers. Further purificationby gravity column chromatography (5% Et2O: hexanes) delivered 34 mg oftrans-3a: Rf0.38 (10% Et2O: hexanes; cis-3a: Rf0.29); IR (thin film)1824 cm−1; 1H NMR (500 MHz, CDCl3) δ 4.22 (ddd, J=4.2, 6.0, 7.5 Hz, 1H),3.16 (ddd, J=3.9, 6.6, 9.0 Hz, 1H), 1.67-1.93 (m, 4H), 1.26-1.49 (m,10H), 0.91 (bs, 6H); 13C NMR (300 MHz, CDCl3): 14.5, 14.7, 23.1, 23.2,25.4, 28.3, 29.9, 32.1, 35.2, 56.9, 78.9, 172.5; ESI LRMS Calcd forC12H22O2 [M+Li]: 205. Found: 205.

(3R, 4S)-3-butyl-3-methyl-4-pentyloxetan-2-one (8a)

A solution of β-lactone 3a (36.3 mg, 0.1835 mmol) in 1.9 mL THF wascooled to −78° C. and 370 μL of LiHMDS (0.367 mmol, 2.0 equiv, 1.0 Msolution in THF) was added under a nitrogen atmosphere. After 1.5 h, 23μL (0.367 mmol, 2.0 equiv) of iodomethane was added and the reactionwarmed to −40° C. and stirred for an additional 45 min. The reactionmixture was concentrated in vacuo and purified by flash chromatography(0→15% Et2O: hexanes) to give β-lactone 8a (28.4 mg, 73%) as a colorlessoil and as a mixture of cis/trans diastereomers (dr, 6:1). Data providedfor major diastereomer: Rf(15% Et2O:hexanes) 0.64; IR (thin film) 1824cm−1; 1H NMR (500 MHz, CDCl3) δ 4.18 (dd (major diast.), J=6.0, 8.5Hz,1H), 1.63-1.78 (m, 2H), 1.45-1.53 (m, 2H), 1.39 (s, 3H), 1.24-1.39 (m,10H), 0.89-0.94 (m, 6H); 13C NMR (500 MHz, CDCl3) 14.1, 14.2, 20.0,22.1, 25.5, 25.6, 26.5, 30.3, 31.7, 35.9, 56.8, 84.5, 175.5; ESI LRMSCalcd. for C13H24O2Li [M+Li]: 219. Found: 219.

(3S, 4S)-3-benzl-3-butyl-4-pentyloxetan-2-one

To a −78° C. solution of β-lactone 3a (153 mg, 0.76 mmol) dissolved in7.5 mL THF was added 1.52 mL LiHMDS (1.52 mmol, 2.0 equiv, 1.0 Msolution in THF) under nitrogen atmosphere. After 1.5 h, 180 μL (1.52mmol, 2.0 equiv) of benzyl bromide was added and the reaction warmed to−40° C. and stirred for an additional 45 min. The reaction mixture wasconcentrated in vacuo and purified by flash chromatography (0→5% Et2O:hexanes) to give β-lactone 8b (192 mg, 88%) as a mixture of cis/transdiastereomers (>19:1). Data provided for major diastereomer: Rf 0.55(5%Et2O:Hexanes); IR (thin film) 1813 cm−1; 1H NMR (500 MHz, CDCl13) δ7.30-7.34 (m, 2H), 7.25-7.28 (m, 1H), 7.16-7.17 (m, 2H), 4.34 (dd,J=4.5, 9.5 Hz, 1H), 3.13, 2.88 (AB q, J=14.5 Hz, 2H), 1.68-1.78 (m, 2H),1.45-1.61 (m, 4H), 1.18-1.39 (m, 8H), 0.94 (t, J=3.5, 3H), 0.87 (t,J=3.5, 3H); 13C NMR (500 MHz, CDCl3): 13.87, 13.89, 22.4, 23.2, 25.2,26.3, 28.7, 29.7, 31.4, 38.2, 61.3, 80.3, 127.1 (2C), 128.7 (2C), 129.8,135.8, 174.2; ESI LRMS Calcd. for C19H28O2 [M+Li]: 295. Found: 295.

(3S,4S)-benzyl 3-butyl-2-oxo-4-pentyloxetane-3-carboxylate (8c)

To a −78° C. solution of NaHMDS (1.2 equiv, 0.91 mmol, 45 μL, 2M in THF)in 5 mL THF was added 150 mg (0.73 mmol) of β-lactone 3a dissolved in2.5 mL THF. After 1.5 h, benzylchloroformate (1.1 equiv, 0.833 mmol, 120μL) was added at one time and stirred for an additional 3 h. Thissolution was warmed to 23° C. over 1 h and worked up as described abovefor β-lactone 8b. Flash chromatography on SiO2 (5 % Et2O: hexanes) gaveβ-lactone 8c (185 mg, 74% yield) as a colorless oil: Rf 0.48 (15% Et2O :Hexanes ); IR (thin film) 1824, 1757, 1716 cm−1; 1H NMR (300 MHz, CDCl3)δ 7.40 (m, 5H), 5.27, 5.22 (AB q, J=12.0, 2H), 4.52 (dd, J=4.8, 8.4 Hz,11H), 2.50-2.56 (m, 1H), 2.22-2.30 (m, 2H), 1.71-1.85 (m, 1H), 1.44-1.60(m, 4H), 1.15-1.39 (m, 8H), 0.81-0.92 (m, 6H); 13C NMR (500 MHz, CDCl3)14, 14.6, 14.7, 23.1, 23.3, 23.6, 24.9, 25.8, 26.9, 29.4, 30.4, 31.1,31.9, 32.3, 40.4, 71.6, 79.8, 121.7, 129.2, 129.4, 129.5, 129.8, 133.1,152.7, 163.7, 167.3; ESI LRMS Calcd. for C20H28LiO4+[M+Li]:339. Found:338.

Example V Screening Beta-Lactones for Activity Against Fatty AcidSynthase and Anti-tumor Activity

Compounds that have been synthesized using the invention synthesismethod are tested for potency and selectivity in their ability to act asagonists of Fatty Acid Synthase (FAS) and its recombinant thioesterasedomain. Based on the structure of Orlistat, it is determined that FASmakes two dominant contacts with the compound: one engaging the longbeta-hydrophilic alkyl chain; and another that forms hydrogen bonds withthe N-formyl leucine.

Enzyme Inhibition Studies.

A recombinant form of the thioesterase domain of fatty acid synthase wasused in a substrate-based screen to measure the apparent K.sub.i ofthese greatly simplified Orlistat derivatives. Importantly, Orlistat isan irreversible inhibitor of the FAS TE domain, as it forms an adductwith the active site serine. We presume that the beta-lactones discussedherein function via the same mechanism. Therefore, we report the resultsas apparent inhibition constants (app K.sub.i), as the term K.sub.i isusually used for reversible inhibitors.

4-Methylumbelliferyl heptanoate (4-MUH) was utilized as a substrate forFAS as it was found to provide the best signal to noise ratio and anacceptable turnover rate. The product of this substrate,4-methylumbelliferone, fluoresces at 450 nM (excitation at 350 nM),providing a convenient readout of thioesterase activity. In this assay,the hydrolysis of 4-MUH is blocked by Orlistat, our lead antagonist withan apparent K.sub.i of 0.21 micro.M. The ability of the simpledialkyl-beta-lactones prepared by the ketene dimerization/hydrogenationprocess to act as antagonists against 4-MUH against recombinant FAS TEwas measured by this assay (Table 5).

TABLE 5 Antagonistic activity of beta-lactones toward recombinant FAS TEcompared to Orlistat. cmpd. R apparent K_(i) (micro.M) cmpd. R apparentK_(i) (micro.M)

2a

16.7 Orlistat 2c

>100 3a

23.1 ± 4.2 2d

5.04 ± 2.1 3b

13.5 ± 7.8 2f

20.8 ± 1.4 3c

 7.7 ± 0.43 3d

 2.5 ± 0.47 3d

>100 3f

35.4 ± 5.3

trans-3a

4.01 ± 1.9 8a CH₃ 14.6 ± 4.0 trans-3b

 6.7 ± 1.0 8b PhCH₂  19.2 ± 0.36 trans-3c

 6.3 ± 0.71 8c CO₂Benzyl 9.96 ± 2.8 trans-3d

 3.3 ± 0.2 trans-3f

These simple dialkyl-beta-lactones are devoid of the aminoester sidechain, which presumably serves as a recognition site and add some degreeof water solubility. Nonetheless, the tested beta-lactones were able toinhibit FAS TE, and, in comparison to Orlistat, the potency of some ofthese compounds was only reduced by approximately 10-fold. (e.g., FIG.13) Thus, these findings are very significant with respect tosynthesizing active beta-lactone compounds using the current inventionmethod.

Thus, there is herein described a scaleable methodology to prepare cisand trans-3,4-disubstituted-beta-lactones using a ketenedimerization/hydrogenation sequence from readily available acidchlorides in good overall yields and high enantioselectivity. Thisreaction could be run as a single-pot, two step process, but higheroverall yields and optical purities were obtained upon isolation of theketene dimer by silica gel chromatography and subsequent hydrogenationof the purified dimer at moderate pressures. Ketene dimers were found tobe isolable and could be purified and their enantiomeric puritydetermined by chiral GC analysis. Enolization followed by alkylation andacylation of the cis-beta-lactones providing ready access totrisubstituted-beta-lactones in high diastereoselectivities.Trans-beta-lactones could be obtained by low temperature deprotonationand quenching. These highly simplified dialkyl-beta-lactones areanalogous to Orlistat and were found to exhibit moderate inhibitoryactivity toward recombinant FAS TE as measured by enzymatic activityusing recombinant protein and a fluorogenic assay. Furthertransformations of these optically pure ketene homodimers are beingexplored as a means to provide practical routes to beta-lactones morestructurally analogous to Orlistat and expected to have higher affinityfor FAS TE.

Methods

Fluorogenic Assay for Detection of Enzyme Inhibition.

Expression of the recombinant thioesterase domain of FAS was performedas described previously (Kridel, S. J.; Axelrod, F.; Rozenkrantz, N.;Smith, J. W. Cancer Res. 2004, 64, 2070 and Knowles, L. M.; Axelrod, F.;Browne, C. D.; Smith, J. W. J. Biol. Chem. 2004, 279, 30540) andlarge-scale expression was preformed by Invitrogen Corporation (Madison,Wis.). The synthetic fluorogenic substrate, 4-methylumbelliferylheptanoate (4-MUH), was purchased from Sigma (St. Louis, Mo.). Thereaction mixture consisted of 500 nM FAS TE in buffer A (100 mMTris-HCl, 50 mM NaCl at pH 7.4) which was pre-incubated with 2.5 .micro.L test beta-lactones dissolved in DMSO at concentrations of0.32-100 . micro.M at 37 . deg.C. for 30 minutes. The reaction wasinitiated by addition of 5 .micro.L of 1.25 mM 4-MUR in 1:1 DMSO:bufferA. The resulting fluorescence from liberated 4-methylumbelliferone wasmeasured every five minutes at 350/450 nm for 40-60 minutes. Results arethe average of triplicate time points. Each compound was tested at leasttwice, yielding essentially identical results.

Incorporation by Reference

Throughout this application, various publications, patents, and/orpatent applications are referenced in order to more fully describe thestate of the art to which this invention pertains. The disclosures ofthese publications, patents, and/or patent applications are hereinincorporated by reference in their entireties, and for the subjectmatter for which they are specifically referenced in the same or a priorsentence, to the same extent as if each independent publication, patent,and/or patent application was specifically and individually indicated tobe incorporated by reference.

Other Embodiments

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1. A method for the asymmetric synthesis of beta-lactone compoundscomprising the steps of: (a) forming a ketene dimer from an acidchloride, in the presence of a catalyst, wherein the catalyst isselected from the group consisting of QND, O-TBS QND, O-TMS QND, andO-TMS QUIN; and (b) hydrogenating the ketene dimer to generate acis-beta-lactone in the presence of a catalyst.
 2. The method of claim 1wherein the beta-lactone synthesis method is a one-pot synthesis method.3. The method of claim 1 wherein the ketene dimer formed in step (a) isisolated before the hydrogenation step.
 4. The method of claim 3 whereinthe isolation of the ketene dimer is performed by silica gelpurification.
 5. The method of claim 1 wherein the acid chloride has theformula VII below

wherein R comprises a hydrogen, an alkyl group, a cycloalkyl group, aheterocycloalkyl group, an alkoxy group, a halogenated alkyl group, analkoxyalkyl group, an alkenyl group, an alkynyl group, an aryl group, aheteroaryl group, an aralkyl group, an amide, an ester, a carbonategroup, a carboxylic acid, an aldehyde, a keto group, an ether group, ahalide, a urethane group, a silyl group or a sulfo-oxo group.
 6. Themethod of claim 5 wherein R comprises n-butyl, cyclopentyl, cyclohexyl,benzyl, CH.sub.2CO.sub.2Me or 11-methoxynonyl.
 7. The method of claim 6wherein R is n-butyl.
 8. The method of claim 1 wherein the step ofhydrogenating the ketene dimer comprise using a palladium on carboncatalyst.
 9. The method of claim 8 wherein the catalyst is at aconcentration of about between 1 mol % and 5 mol %.
 10. The method ofclaim 9 wherein the catalyst is at a concentration of about 1 mol %. 11.The method of claim 8 what the catalyst is accompanied by an amine. 12.The method of claim 11 wherein the mine is triethylamine.
 13. The methodof claim 8 wherein the step of hydrogenating a ketene dimer is performedfor 30 minutes at 30 psi H.sub.2.
 14. The method of claim 1 furthercomprising an epimerization step.
 15. The method of claim 14 wherein theepimerization step comprises the further steps of: (a) deprotonating thecis-beta lactone; and (b) quenching the deprotonated species under lowtemperature.
 16. The method of claim 15 wherein the step ofdeprotonating is performed using lithium hexamethyldisilazide (LiHMDS)or related bases such as lithium diisopropylamide (LDA), sodiumhexamethyldisilazide (NaHMDS), or lithium tetramethylpiperidide (LiTMP).17. The method of claim 15 wherein the step of quenching thedeprotonated species is performed using acetic acid.
 18. The method ofclaim 1 further comprising converting the cis-beta-lactone to atrisubstituted species by the further steps of: (a) enolization of thecis-beta-lactone; and (b) addition of electrophiles.
 19. The method ofclaim 18 wherein the converting step comprises alkylation or acylation.20. The method of claim 18 wherein the enolization step is performedusing LDA, LiHMDS or NaHMDS.
 21. The method of claim 18 wherein theelectrophile comprises methyl iodide, benzyl or CO.sub.2benzyl.